Adeno-associated virus-mediated delivery of GDNF to skeletal muscles

ABSTRACT

Compositions and methods for delivering GDNF to skeletal muscles to result in a therapeutic effect are disclosed. The compositions and methods use adeno-associated virus (AAV)-based gene delivery systems. The methods are useful for treating motoneuron diseases, such as amyotrophic lateral sclerosis (ALS).

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 10/327,620, filed Dec. 19, 2002 from which priorityis claimed pursuant to 35 U.S.C. §120, which application claims thebenefit of provisional patent application Ser. No. 60/342,304, filedDec. 19, 2001 under 35 U.S.C. §119(e), which applications are herebyincorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates generally to compositions and methods forgene delivery. In particular, the present invention pertains toadeno-associated virus (AAV)-based gene delivery systems for deliveringglial cell line-derived neurotrophic factor (GDNF) to skeletal muscle totreat motoneuron diseases such as amyotrophic lateral sclerosis (ALS).

BACKGROUND OF THE INVENTION

Motoneuron neurodegenerative diseases present major public healthissues. For example, amyotrophic lateral sclerosis (ALS) is arelentlessly progressive lethal disease that involves selectiveannihilation of motoneurons. Approximately 20% of familial ALS is linkedto mutations in the Cu/Zn superoxide dismutase (SOD1) gene (Julien, J.P., Cell (2001) 104:581-591). Transgenic mice overexpressing this mutantgene (mSOD1G93A) develop a dominantly inherited adult-onset paralyticdisorder that has many of the clinical and pathological features offamilial ALS (Gurney et al., Science (1994) 264:1772-1775). However, todate, the molecular mechanisms leading to motoneuron degeneration in ALSand most motor neuron diseases remain poorly understood. Because themechanism leading to motoneuron degeneration in ALS is not known, thereis currently no therapy available to prevent or cure ALS.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to bethe most potent neurotrophic factor for the proliferation,differentiation, and survival of spinal motoneurons. GDNF and GDNF MRNAlevels have been reported to be up-regulated in denervated muscles asfound in ALS, polymyostits (PM) and muscular dystrophy (MD), or afterperipheral nerve lesion and the like (Trupp et al., J Cell Biol. (1995)130:137-148; Lie and Weis, Neurosci. Lett. (1998) 250: 87-90; Yamamotoet al., Neurochem. Res. (1999) 24:785-790). GDNF has been proposed as atherapeutic agent to treat motor neuron disease (Henderson et al.,Science (1994) 266:1062-1064; Oppenheim et al., Nature (1995)373:344-346; Yan et al., Nature (1995) 373:341-344; Sagot et al., JNeurosci. (1996) 16: 2335-2341; Bohn, M. C., Biochem. Pharmacol. (1999)57:135-142; Mohajeri et al., Hum. Gene Ther. (1999) 10:1853-1866).Neurotrophic factors such as GDNF have been shown to slow motoneurondegeneration and to restore the function of non-functional motoneuronsthat are still alive (Trupp et al., J. Cell Biol. (1995) 130:137-148;Sagot et al., J Neurosci. (1998) 18: 1132-1141; Lie and Weis, Neurosci.Lett. (1998) 250: 87-90; Baumgartner and Shine, J. Neurosci. Res. (1998)54: 766-777; Yamamoto et al., Neurochem. Res. (1999) 24:785-790; Bieschand Tuszynski, J. Comp. Neurol. (2001) 436:399-410; Keller-Peck et al.,J Neurosci. (2001) 21:6136-6146.

To date, however, clinical trials using repeated administration ofrecombinant GDNF, as well as other neurotrophic factors such as ciliaryneurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) andinsulin-like growth factor-I (IGF-I), have shown limited or no promiseand/or have resulted in severe side-effects (Yuen, E. C., Phys. Med.Rehabil. Clin. N. Am. (2001) 12:293-306). In particular, these proteinshave a short in vivo plasma half-life, have poor access to spinal cordmotoneurons, and cause inflammatory reactions that preventadministration at an adequate dose (Haase et al., Nat. Med.(1997)3:429-436; Alisky and Davidson, Hum. Gene Ther. (2000)11:2315-2329). These limitations, together with the chronicity andprogressive nature of most motoneuron degenerative diseases, underscorethe necessity to develop innovative strategies that offer more effectiveand long-term delivery of neurotrophic factors to motoneurons. In anattempt to overcome the above-described problems, experimenters havestudied gene-therapy approaches for treating ALS (Alisky and Davidson,Hum. Gene Ther. (2000) 11:2315-2329). Genetically modifiedmyoblast-based GDNF gene delivery in muscles prevented loss of spinalmotoneurons and delayed the onset of the disease in a transgenic mousefamilial ALS model (Mohajeri et al., Hum. Gene Ther. (1999)10:1853-1866). However, the level of GDNF protein in treated muscle wasundetectable.

Adeno-associated virus (AAV) has shown promise for delivering genes forgene therapy in clinical trials in humans (see, e.g., Kay et al., Nat.Genet. (2000) 24:257-261). As the only viral vector system based on anonpathogenic and replication-defective virus, recombinant AAV virionshave been successfully used to establish efficient and sustained genetransfer of both proliferating and terminally differentiated cells in avariety of tissues (Bueler, H., Biol. Chem. (1999) 380:613-622).Notwithstanding these successes, AAV-mediated GDNF gene therapy fortreating motor neuron disease, such as ALS, has not been demonstrated.

The AAV genome is a linear, single-stranded DNA molecule containingabout 4681 nucleotides. The AAV genome generally comprises an internalnonrepeating genome flanked on each end by inverted terminal repeats(ITRs). The ITRs are approximately 145 base pairs (bp) in length. TheITRs have multiple functions, including as origins of DNA replication,and as packaging signals for the viral genome. The internal nonrepeatedportion of the genome includes two large open reading frames, known asthe AAV replication (rep) and capsid (cap) genes. The rep and cap genescode for viral proteins that allow the virus to replicate and packageinto a virion. In particular, a family of at least four viral proteinsare expressed from the AAV rep region, Rep 78, Rep 68, Rep 52, and Rep40, named according to their apparent molecular weight. The AAV capregion encodes at least three proteins, VPI, VP2, and VP3.

AAV has been engineered to deliver genes of interest by deleting theinternal nonrepeating portion of the AAV genome (i.e., the rep and capgenes) and inserting a heterologous gene between the ITRs. Theheterologous gene is typically functionally linked to a heterologouspromoter (constitutive, cell-specific, or inducible) capable of drivinggene expression in the patient's target cells under appropriateconditions. Termination signals, such as polyadenylation sites, can alsobe included.

AAV is a helper-dependent virus; that is, it requires coinfection with ahelper virus (e.g., adenovirus, herpesvirus or vaccinia), in order toform AAV virions. In the absence of coinfection with a helper virus, AAVestablishes a latent state in which the viral genome inserts into a hostcell chromosome, but infectious virions are not produced. Subsequentinfection by a helper virus “rescues” the integrated genome, allowing itto replicate and package its genome into an infectious AAV virion. WhileAAV can infect cells from different species, the helper virus must be ofthe same species as the host cell. Thus, for example, human AAV willreplicate in canine cells coinfected with a canine adenovirus.

However, prior to the present invention, AAV-mediated delivery of GDNFfor the treatment of motoneuron diseases, such as ALS, has not beensuccessfully achieved.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a potent and effectivemethod for treating neurodegenerative diseases that affect motoneurons.As shown herein, skeletal muscle is particularly useful for AAV-mediatedGDNF delivery. Skeletal muscle is highly transducible, easily accessibleand displays a low cell turnover (Wang et al., Proc. Natl. Acad. Sci.USA (2000) 97:13714-13719; Xiao et al., J. Virol. (1996) 70:8098-8108).Nerve terminals are only in contact with myofibers at the neuromuscularjunctions (NMJs) at which barriers against various substances is absent,allowing them to reach the central nervous system. According to theneurotrophic theory, neurites connect with their targets to gain accessto target-derived neurotrophic factors for neuron survival. As atarget-derived neurotrophic factor, endogenous GDNF produced by skeletalmuscle functions via retrograde axonal transport from the target muscletissue to motoneuronal cell bodies in the spinal cord (Mitsumoto, H.,Muscle Nerve (1999) 22:1000-1021). The inventors herein have discoveredthat AAV-mediated GDNF gene delivery via intramuscular administrationdrives substantial and persistent expression of GDNF in large numbers ofmyofibers. Moreover, expressed GDNF is retrogradely transported tospinal cord motoneurons from nerve terminals in the muscle.Significantly, the inventors herein demonstrate through studies in ALSanimal models that AAV-mediated GDNF delivery via skeletal musclesignificantly delays the onset of disease, lengthens the life-span,abates behavioral impairment, and promotes motoneuron survival.

Accordingly, in one embodiment, the invention is directed to a method ofdelivering a recombinant AAV virion to a muscle cell or muscle tissue ofa mammalian subject with a motoneuron disorder. The method comprises:

(a) providing a recombinant AAV virion which comprises a polynucleotideencoding a GDNF operably linked to control elements capable of directingthe in vivo transcription and translation of the GDNF; and

(b) delivering the recombinant AAV virion directly into the muscle cellor muscle tissue of the subject, whereby the GDNF is expressed at alevel which provides a therapeutic effect in the mammalian subject.

In certain embodiments, the muscle cell or tissue is derived fromskeletal muscle. In yet additional embodiments, the recombinant AAVvirion is introduced into the muscle cell in vivo, e.g., byintramuscular injection. In alternative embodiments, the recombinant AAVvirion is introduced into the muscle cell in vitro. In yet furtherembodiments, the motoneuron disorder is amyotrophic lateral sclerosis(ALS).

In another embodiment, the invention is directed to a method ofdelivering a recombinant AAV virion to a skeletal muscle of a humansubject with ALS. The method comprises:

(a) providing a recombinant AAV virion that comprises a polynucleotideencoding a human GDNF operably linked to control elements capable ofdirecting the in vivo transcription and translation of said GDNF; and

(b) delivering the recombinant AAV virion directly into skeletal muscleof the subject in vivo, whereby the GDNF is expressed at a level whichprovides a therapeutic effect in the human subject.

In yet another embodiment, the invention is directed to a method oftreating a mammalian subject with a motoneuron disorder comprisingadministering intramuscularly to the subject recombinantadeno-associated virus (AAV) virions comprising a polynucleotideencoding a GDNF polypeptide operably linked to expression controlelements capable of directing the in vivo transcription and translationof the GDNF to provide a therapeutic effect.

In certain embodiments, the motoneuron disorder is ALS. In additionalembodiments, the subject is human and the polynucleotide encodes a humanGDNF, such as human pre-pro-GDNF. Additionally, the control elements cancomprise a viral promoter, such as an MLP, CMV, or RSV LTR promoter. Inyet additional embodiments, muscle cells are transduced in vivo, e.g.,by administration into skeletal muscle.

In another embodiment, the invention is directed to a method of treatinga mammalian subject with ALS. The method comprises administering intoskeletal muscle of the subject a composition comprising recombinantadeno-associated virus (AAV) virions that comprise a polynucleotideencoding a GDNF polypeptide operably linked to expression controlelements capable of directing the in vivo transcription and translationof the GDNF, to provide a therapeutic effect.

In certain embodiments, the subject is a human and the polynucleotideencodes a human GDNF, such as human pre-pro-GDNF. Additionally, thecontrol elements can comprise a viral promoter, such as an MLP, CMV, orRSV LTR promoter.

These and other embodiments of the subject invention will readily occurto those of skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the GDNF levels in conditioned medium and 293 celllysate 48 post infection with AAV-GDNF-FLAG or AAV-LacZ as measured byELISA.

FIG. 2 shows GDNF levels in gastrocnemius muscle of injected mice atvarious time points post-injection.

FIG. 3 displays percent distribution of muscle fibers of variousdiameters in wild-type, control ALS and AAV-GDNF-treated ALS mice.

FIGS. 4A-4B show the numbers of spinal motoneurons in wild-type, controlALS and AAV-GDNF-treated ALS mice. FIG. 4A shows the average number ofNissl-stained neurons per anterior horn. FIG. 4B shows the averagenumber of SMI-32-stained neurons per anterior horn.

FIGS. 5A and 5B display the effect of GDNF on motoneurons that retainedaxonal projections in wild-type, control ALS and AAV-GDNF-treated ALSmice. FIG. 5A shows the survival of CTB-labeled motoneurons per anteriorhorn. The value represents the CTB/Nissl ratio (average number ofneurons per anterior horn). FIG. 5B shows the percent distribution ofmuscle fibers of various diameters in wild-type, control ALS andAAV-GDNF-treated ALS mice.

FIGS. 6A-6E show the results of experiments demonstrating that GDNFdelays the onset of disease, improves motor performance, and prolongssurvival in transgenic ALS mice. FIG. 6A displays the cumulativeprobability of onset of rotarod deficits in ALS mice. FIG. 6B showsperformance of ALS mice in the rotarod test at 5 rpm. FIG. 6C showsperformance of ALS mice in the rotarod test at 10 rpm. FIG. 6D showsperformance of ALS mice in the rotarod test at 20 rpm. FIG. 6E shows thecumulative probability of survival.

FIG. 7 (SEQ ID NOS: 1 and 2) shows the nucleotide sequence and aminoacid sequence for a human pre-pro-GDNF. The mature GDNF molecule spansamino acid positions 78-211.

FIG. 8 (SEQ ID NOS:3 and 4) shows the nucleotide sequence and amino acidsequence for a rat pre-pro-GDNF. The mature GDNF molecule spans aminoacid positions 78-211.

FIG. 9 (SEQ ID NOS:6 and 7) shows the nucleotide sequence and amino acidsequence for a mouse pre-pro-GDNF. The mature GDNF molecule spans aminoacid positions 107-240.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of virology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (Current Edition); DNACloning: A Practical Approach, Vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition); CRC Handbook of Parvoviruses, vol. I & II (P. Tijssen, ed.);Fundamental Virology, 2^(nd) Edition, vol. I & II (B. N. Fields and D.M. Knipe, eds.); Freshney Culture of Animal Cells, A Manual of BasicTechnique (Wiley-Liss, Third Edition); and Ausubel et al. (1991) CurrentProtocols in Molecular Biology (Wiley Interscience, NY).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

A. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

As used herein, the term “glial cell line-derived neurotrophic factorpolypeptide” or “GDNF polypeptide” refers to a neurotrophic factor ofany origin, which is substantially homologous and functionallyequivalent to any of the various known GDNFs. Representative GDNFs forthree mammalian species are shown in FIGS. 7, 8 and 9. The degree ofhomology between the rat (FIG. 8, SEQ ID NO:4) and human (FIG. 7, SEQ IDNO:2) protein is about 93% and all mammalian GDNFs have a similarly highdegree of homology. Such GDNFs may exist as monomers, dimers or othermultimers in their biologically active form. Thus, the term “GDNFpolypeptide” as used herein encompasses active monomeric GDNFs, as wellas active multimeric GDNFs, active glycosylated and non-glycosylatedforms of GDNF and active truncated forms of the molecule.

By “functionally equivalent” as used herein, is meant a GDNF polypeptidethat retains some or all of the biological properties regardingmotoneurons, but not necessarily to the same degree, as a native GDNFmolecule.

“Homology” refers to the percent similarity between two polynucleotideor two polypeptide moieties. Two polynucleotide, or two polypeptidesequences are “substantially homologous” to each other when thesequences exhibit at least about 50%, preferably at least about 75%,more preferably at least about 80%-85%, preferably at least about 90%,and most preferably at least about 95%-99% or more sequence similarityor sequence identity over a defined length of the molecules. As usedherein, substantially homologous also refers to sequences showingcomplete identity to the specified polynucleotide or polypeptidesequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying the result by 100.

Readily available computer programs can be used to aid in the analysisof similarity and identity, such as ALIGN, Dayhoff, M.O. in Atlas ofProtein Sequence and Structure M. O. Dayhoff ed., 5 Suppl. 3:353-358,National biomedical Research Foundation, Washington, D.C., which adaptsthe local homology algorithm of Smith and Waterman Advances in Appl.Math. 2:482-489, 1981 for peptide analysis. Programs for determiningnucleotide sequence similarity and identity are available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAPprograms, which also rely on the Smith and Waterman algorithm. Theseprograms are readily utilized with the default parameters recommended bythe manufacturer and described in the Wisconsin Sequence AnalysisPackage referred to above. For example, percent similarity of aparticular nucleotide sequence to a reference sequence can be determinedusing the homology algorithm of Smith and Waterman with a defaultscoring table and a gap penalty of six nucleotide positions.

Another method of establishing percent similarity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequencesimilarity.” Other suitable programs for calculating the percentidentity or similarity between sequences are generally known in the art,for example, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

By “GDNF variant” is meant a biologically active derivative of thereference GDNF molecule, or a fragment of such a derivative, thatretains desired activity, such as neurotrophic activity in the assaysdescribed herein. In general, the term “variant” refers to compoundshaving a native polypeptide sequence and structure with one or moreamino acid additions, substitutions (generally conservative in nature)and/or deletions, relative to the native molecule, so long as themodifications do not destroy neurotrophic activity. Preferably, thevariant has at least the same biological activity as the nativemolecule. Methods for making polynucleotides encoding for GDNF variantsare known in the art and are described further below.

For GDNF deletion variants, deletions generally range from about 1 to 30residues, more usually from about 1 to 10 residues, and typically fromabout 1 to 5 contiguous residues, or any integer within the statedranges. N-terminal, C-terminal and internal deletions are contemplated.Deletions are generally introduced into regions of low homology withother TGF-β super family members in order to preserve maximum biologicalactivity. Deletions are typically selected so as to preserve thetertiary structure of the GDNF protein product in the affected domain,e.g., cysteine crosslinking. Non-limiting examples of deletion variantsinclude truncated GDNF protein products lacking from 1-40 N-terminalamino acids of GDNF, or variants lacking the C-terminal residue of GDNF,or combinations thereof.

For GDNF addition variants, amino acid sequence additions typicallyinclude N-and/or C-terminal fusions ranging in length from one residueto polypeptides containing a hundred or more residues, as well asinternal additions of single or multiple amino acid residues. Internaladditions generally range from about 1-10 residues, more typically fromabout 1-5 residues, and usually from about 1-3 amino acid residues, orany integer within the stated ranges. Examples of N-terminal additionvariants include the fusion of a heterologous N-terminal signal sequenceto the N-terminus of GDNF as well as fusions of amino acid sequencesderived from the sequence of other neurotrophic factors.

GDNF substitution variants have at least one amino acid residue of theGDNF amino acid sequence removed and a different residue inserted in itsplace. Such substitution variants include allelic variants, which arecharacterized by naturally occurring nucleotide sequence changes in thespecies population that may or may not result in an amino acid change.Particularly preferred substitutions are conservative in nature, i.e.,those substitutions that take place within a family of amino acids thatare related in their side chains. Specifically, amino acids aregenerally divided into four families: (1) acidic—aspartate andglutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine,cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, andtyrosine are sometimes classified as aromatic amino acids. For example,it is reasonably predictable that an isolated replacement of leucinewith isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar conservative replacement of an amino acidwith a structurally related amino acid, will not have a major effect onthe biological activity.

For example, the GDNF molecule may include up to about 5-10 conservativeor non-conservative amino acid substitutions, or even up to about 15-25conservative or non-conservative amino acid substitutions, or anyinteger between 5-25, so long as the desired function of the moleculeremains intact. One of skill in the art may readily determine regions ofthe molecule of interest that can tolerate change using techniques wellknown in the art.

Specific mutations of the GDNF amino acid sequence may involvemodifications to a glycosylation site (e.g., serine, threonine, orasparagine). The absence of glycosylation or only partial glycosylationresults from amino acid substitution or deletion at anyasparagine-linked glycosylation recognition site or at any site of themolecule that is modified by addition of an O-linked carbohydrate. Anasparagine-linked glycosylation recognition site comprises a tripeptidesequence which is specifically recognized by appropriate cellularglycosylation enzymes. These tripeptide sequences are either Asn-Xaa-Thror Asn-Xaa-Ser, where Xaa can be any amino acid other than Pro. Avariety of amino acid substitutions or deletions at one or both of thefirst or third amino acid positions of a glycosylation recognition site(and/or amino acid deletion at the second position) result innon-glycosylation at the modified tripeptide sequence. Thus, theexpression of appropriate altered nucleotide sequences produces variantswhich are not glycosylated at that site. Alternatively, the GDNF aminoacid sequence may be modified to add glycosylation sites.

Methods for identifying GDNF amino acid residues or regions formutagenesis are well known in the art. One such method is known as“alanine scanning mutagenesis.” See, e.g., Cunningham and Wells, Science(1989) 244:1081-1085. In this method, an amino acid residue or group oftarget residues are identified (e.g., charged residues such as Arg, Asp,His, Lys, and Glu) and replaced by a neutral or negatively charged aminoacid (most preferably alanine or polyalanine) to affect the interactionof the amino acids with the surrounding aqueous environment in oroutside the cell. Those domains demonstrating functional sensitivity tothe substitutions are refined by introducing additional or alternateresidues at the sites of substitution. Thus, the target site forintroducing an amino acid sequence variation is determined, alaninescanning or random mutagenesis is conducted on the corresponding targetcodon or region of the DNA sequence, and the expressed GDNF variants arescreened for the optimal combination of desired activity and degree ofactivity.

The sites of greatest interest for mutagenesis include sites where theamino acids found in GDNF proteins from various species aresubstantially different in terms of side-chain bulk, charge, and/orhydrophobicity. Other sites of interest are those in which particularresidues of GDNF-like proteins, obtained from various species, areidentical. Such positions are generally important for the biologicalactivity of a protein. Initially, these sites are substituted in arelatively conservative manner. If such substitutions result in a changein biological activity, then more substantial changes (exemplarysubstitutions) are introduced, and/or other additions or deletions maybe made, and the resulting products screened for activity.

Assays for GDNF activity are known in the art. For example, any of thevarious in vitro model systems, described more fully below, can be usedas measures of GDNF activity.

By “motoneuron disorder” is meant a disease affecting a neuron withmotor function, i.e., a neuron that conveys motor impulses. Such neuronsare also termed “motor neruons.” These neurons include, withoutlimitation, alpha neurons of the anterior spinal cord that give rise tothe alpha fibers which innervate the skeletal muscle fibers; betaneurons of the anterior spinal cord that give rise to the beta fiberswhich innervate the extrafusal and intrafusal muscle fibers; gammaneurons of the anterior spinal cord that give rise to the gamma(fusimotor) fibers which innervate the intrafusal fibers of the musclespindal; heteronymous neurons that supply muscles other than those fromwhich afferent impulses originate; homonymous neurons that supplymuscles from which afferent impulses originate; lower peripheral neuronswhose cell bodies lie in the ventral gray columns of the spinal cord andwhose terminations are in skeletal muscles; peripheral neurons thatreceive impulses from intemuerons; and upper neurons in the cerebralcortex that conduct impulses from the motor cortex to motor nuclei ofthe cerebral nerves or to the ventral gray columns of the spinal cord.

Nonlimiting examples of motoneuron disorders include the variousamyotrophies such as hereditary amyotrophies including hereditary spinalmuscular atrophy, acute infantile spinal muscular atrophy such asWerdnig-Hoffman disease, progressive muscular atrophy in children suchas the proximal, distal type and bulbar types, spinal muscular atrophyof adolescent or adult onset including the proximal, scapuloperoneal,facioscapulohumeral and distal types, amyotrophic lateral sclerosis(ALS) and primary lateral sclerosis (PLS). Also included within the termis motoneuron injury. By “skeletal muscle” is meant a striated musclethat is attached to bones and that typically crosses at least one joint.Scientifically, these muscles are often referred to as musculi skeleti.

By “vector” is meant any genetic element, such as a plasmid, phage,transposon, cosmid, chromosome, virus, virion, etc., which is capable ofreplication when associated with the proper control elements and whichcan transfer gene sequences between cells. Thus, the term includescloning and expression vehicles, as well as viral vectors.

By an “AAV vector” is meant a vector derived from an adeno-associatedvirus serotype, including without limitation, AAV-1, AAV-2, AAV-3,AAV-4, AAV-5, AAV-6, AAV-7 and AAV-8. AAV vectors can have one or moreof the AAV wild-type genes deleted in whole or part, preferably the repand/or cap genes, but retain functional flanking ITR sequences.Functional ITR sequences are necessary for the rescue, replication andpackaging of the AAV virion. Thus, an AAV vector is defined herein toinclude at least those sequences required in cis for replication andpackaging (e.g., functional ITRs) of the virus. The ITRs need not be thewild-type nucleotide sequences, and may be altered, e.g., by theinsertion, deletion or substitution of nucleotides, so long as thesequences provide for functional rescue, replication and packaging.

“AAV helper functions” refer to AAV-derived coding sequences which canbe expressed to provide AAV gene products that, in turn, function intrans for productive AAV replication. Thus, AAV helper functions includeboth of the major AAV open reading frames (ORFs), rep and cap. The Repexpression products have been shown to possess many functions,including, among others: recognition, binding and nicking of the AAVorigin of DNA replication; DNA helicase activity; and modulation oftranscription from AAV (or other heterologous) promoters. The Capexpression products supply necessary packaging functions. AAV helperfunctions are used herein to complement AAV functions in trans that aremissing from AAV vectors.

The term “AAV helper construct” refers generally to a nucleic acidmolecule that includes nucleotide sequences providing AAV functionsdeleted from an AAV vector which is to be used to produce a transducingvector for delivery of a nucleotide sequence of interest. AAV helperconstructs are commonly used to provide transient expression of AAV repand/or cap genes to complement missing AAV functions that are necessaryfor lytic AAV replication; however, helper constructs lack AAV ITRs andcan neither replicate nor package themselves. AAV helper constructs canbe in the form of a plasmid, phage, transposon, cosmid, virus, orvirion. A number of AAV helper constructs and vectors that encode Repand/or Cap expression products have been described. See, e.g., U.S. Pat.Nos. 6,001,650, 5,139,941 and 6,376,237, all incorporated herein byreference in their entireties; Samulski et al. (1989) J Virol.63:3822-3828; and McCarty et al. (1991) J Virol. 65:2936-2945.

The term “accessory functions” refers to non-AAV derived viral and/orcellular functions upon which AAV is dependent for its replication.Thus, the term captures proteins and RNAs that are required in AAVreplication, including those moieties involved in activation of AAV genetranscription, stage specific AAV mRNA splicing, AAV DNA replication,synthesis of Cap expression products and AAV capsid assembly.Viral-based accessory functions can be derived from any of the knownhelper viruses such as adenovirus, herpesvirus (other than herpessimplex virus type-1) and vaccinia virus.

The term “accessory function vector” refers generally to a nucleic acidmolecule that includes nucleotide sequences providing accessoryfunctions. An accessory function vector can be transfected into asuitable host cell, wherein the vector is then capable of supporting AAVvirion production in the host cell. Expressly excluded from the term areinfectious viral particles as they exist in nature, such as adenovirus,herpesvirus or vaccinia virus particles. Thus, accessory fimctionvectors can be in the form of a plasmid, phage, transposon or cosmid.

In particular, it has been demonstrated that the full-complement ofadenovirus genes are not required for accessory helper functions. Inparticular, adenovirus mutants incapable of DNA replication and lategene synthesis have been shown to be permissive for AAV replication. Itoet al., (1970) J Gen. Virol. 9:243; Ishibashi et al, (1971) Virology45:317. Similarly, mutants within the E2B and E3 regions have been shownto support AAV replication, indicating that the E2B and E3 regions areprobably not involved in providing accessory functions. Carter et al.,(1983) Virology 126:505. However, adenoviruses defective in the E1region, or having a deleted E4 region, are unable to support AAVreplication. Thus, E1A and E4 regions are likely required for AAVreplication, either directly or indirectly. Laughlin et al., (1982) JVirol. 41:868; Janik et al., (1981) Proc. Natl. Acad. Sci. USA 78:1925;Carter et al., (1983) Virology 126:505. Other characterized Ad mutantsinclude: E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra;Ostrove et al., (1980) Virology 104:502); E2A (Handa et al., (1975) J.Gen. Virol. 29:239; Strauss et al., (1976) J. Virol. 17:140; Myers etal., (1980) J. Virol. 35:665; Jay et al., (1981) Proc. Natl. Acad. Sci.USA 78:2927; Myers et al., (1981) J Biol. Chem. 256:567); E2B (Carter,Adeno-Associated Virus Helper Functions, in I CRC Handbook ofParvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983), supra);and E4 (Carter et al.(1983), supra; Carter (1995)). Although studies ofthe accessory functions provided by adenoviruses having mutations in theE1B coding region have produced conflicting results, Samulski et al.,(1988) J. Virol. 62:206-210, recently reported that E1B55k is requiredfor AAV virion production, while E1B19k is not. In addition,International Publication WO 97/17458 and Matshushita et al., (1998)Gene Therapy 5:938-945, describe accessory function vectors encodingvarious Ad genes.

Particularly preferred accessory function vectors comprise an adenovirusVA RNA coding region, an adenovirus E4 ORF6 coding region, an adenovirusE2A 72 kD coding region, an adenovirus E1A coding region, and anadenovirus E1B region lacking an intact E1B55k coding region. Suchvectors are described in International Publication No. WO 01/83797.

By “capable of supporting efficient rAAV virion production” is meant theability of an accessory function vector or system to provide accessoryfunctions that are sufficient to complement rAAV virion production in aparticular host cell at a level substantially equivalent to or greaterthan that which could be obtained upon infection of the host cell withan adenovirus helper virus. Thus, the ability of an accessory functionvector or system to support efficient rAAV virion production can bedetermined by comparing rAAV virion titers obtained using the accessoryvector or system with titers obtained using infection with an infectiousadenovirus. More particularly, an accessory function vector or systemsupports efficient rAAV virion production substantially equivalent to,or greater than, that obtained using an infectious adenovirus when theamount of virions obtained from an equivalent number of host cells isnot more than about 200 fold less than the amount obtained usingadenovirus infection, more preferably not more than about 100 fold less,and most preferably equal to, or greater than, the amount obtained usingadenovirus infection.

By “recombinant virus” is meant a virus that has been geneticallyaltered, e.g., by the addition or insertion of a heterologous nucleicacid construct into the particle.

By “AAV virion” is meant a complete virus particle, such as a wild-type(wt) AAV virus particle (comprising a linear, single-stranded AAVnucleic acid genome associated with an AAV capsid protein coat). In thisregard, single-stranded AAV nucleic acid molecules of eithercomplementary sense, e.g., “sense” or “antisense” strands, can bepackaged into any one AAV virion and both strands are equallyinfectious.

A “recombinant AAV virion,” or “rAAV virion” is defined herein as aninfectious, replication-defective virus including an AAV protein shell,encapsidating a heterologous nucleotide sequence of interest which isflanked on both sides by AAV ITRs. A rAAV virion is produced in asuitable host cell which has had an AAV vector, AAV helper functions andaccessory functions introduced therein. In this manner, the host cell isrendered capable of encoding AAV polypeptides that are required forpackaging the AAV vector (containing a recombinant nucleotide sequenceof interest) into infectious recombinant virion particles for subsequentgene delivery.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell, and a cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties, such as a nucleotide integration vector and other nucleicacid molecules, into suitable host cells.

The term “host cell” denotes, for example, microorganisms, yeast cells,insect cells, and mammalian cells, that can be, or have been, used asrecipients of an AAV helper construct, an AAV vector plasmid, anaccessory function vector, or other transfer DNA. The term includes theprogeny of the original cell which has been transfected. Thus, a “hostcell” as used herein generally refers to a cell which has beentransfected with an exogenous DNA sequence. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

As used herein, the term “cell line” refers to a population of cellscapable of continuous or prolonged growth and division in vitro. Often,cell lines are clonal populations derived from a single progenitor cell.It is further known in the art that spontaneous or induced changes canoccur in karyotype during storage or transfer of such clonalpopulations. Therefore, cells derived from the cell line referred to maynot be precisely identical to the ancestral cells or cultures, and thecell line referred to includes such variants.

The term “heterologous” as it relates to nucleic acid sequences such ascoding sequences and control sequences, denotes sequences that are notnormally joined together, and/or are not normally associated with aparticular cell. Thus, a “heterologous” region of a nucleic acidconstruct or a vector is a segment of nucleic acid within or attached toanother nucleic acid molecule that is not found in association with theother molecule in nature. For example, a heterologous region of anucleic acid construct could include a coding sequence flanked bysequences not found in association with the coding sequence in nature.Another example of a heterologous coding sequence is a construct wherethe coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, acell transformed with a construct which is not normally present in thecell would be considered heterologous for purposes of this invention.Allelic variation or naturally occurring mutational events do not giverise to heterologous DNA, as used herein.

A “coding sequence” or a sequence which “encodes” a particular protein,is a nucleic acid sequence which is transcribed (in the case of DNA) andtranslated (in the case of MRNA) into a polypeptide in vitro or in vivowhen placed under the control of appropriate regulatory sequences. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A coding sequence can include, but is not limited to, cDNAfrom prokaryotic or eukaryotic MRNA, genomic DNA sequences fromprokaryotic or eukaryotic DNA, and even synthetic DNA sequences. Atranscription termination sequence will usually be located 3′ to thecoding sequence.

A “nucleic acid” sequence refers to a DNA or RNA sequence. The termcaptures sequences that include any of the known base analogues of DNAand RNA such as, but not limited to 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, -uracil-5-oxyacetic acid methylester, uracil-5-oxyaceticacid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites (“IRES”), enhancers, and the like, which collectivelyprovide for the replication, transcription and translation of a codingsequence in a recipient cell. Not all of these control sequences needalways be present so long as the selected coding sequence is capable ofbeing replicated, transcribed and translated in an appropriate hostcell.

The term “promoter” is used herein in its ordinary sense to refer to anucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence. Transcription promoters can include“inducible promoters” (where expression of a polynucleotide sequenceoperably linked to the promoter is induced by an analyte, cofactor,regulatory protein, etc.), “repressible promoters” (where expression ofa polynucleotide sequence operably linked to the promoter is induced byan analyte, cofactor, regulatory protein, etc.), and “constitutivepromoters”.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

By “isolated” when referring to a nucleotide sequence, is meant that theindicated molecule is present in the substantial absence of otherbiological macromolecules of the same type. Thus, an “isolated nucleicacid molecule which encodes a particular polypeptide” refers to anucleic acid molecule which is substantially free of other nucleic acidmolecules that do not encode the subject polypeptide; however, themolecule may include some additional bases or moieties which do notdeleteriously affect the basic characteristics of the composition.

For the purpose of describing the relative position of nucleotidesequences in a particular nucleic acid molecule throughout the instantapplication, such as when a particular nucleotide sequence is describedas being situated “upstream,” “downstream,” “3′, ” or “5′” relative toanother sequence, it is to be understood that it is the position of thesequences in the “sense” or “coding” strand of a DNA molecule that isbeing referred to as is conventional in the art.

A “functional homologue,” or a “functional equivalent” of a given AAVpolypeptide includes molecules derived from the native polypeptidesequence, as well as recombinantly produced or chemically synthesizedpolypeptides which function in a manner similar to the reference AAVmolecule to achieve a desired result. Thus, a functional homologue ofAAV Rep68 or Rep78 encompasses derivatives and analogues of thosepolypeptides—including any single or multiple amino acid additions,substitutions and/or deletions occurring internally or at the amino orcarboxy termini thereof—so long as integration activity remains.

A “functional homologue,” or a “functional equivalent” of a givenadenoviral nucleotide region includes similar regions derived from aheterologous adenovirus serotype, nucleotide regions derived fromanother virus or from a cellular source, as well as recombinantlyproduced or chemically synthesized polynucleotides which function in amanner similar to the reference nucleotide region to achieve a desiredresult. Thus, a functional homologue of an adenoviral VA RNA gene regionor an adenoviral E2a gene region encompasses derivatives and analoguesof such gene regions—including any single or multiple nucleotide baseadditions, substitutions and/or deletions occurring within the regions,so long as the homologue retains the ability to provide its inherentaccessory function to support AAV virion production at levels detectableabove background.

“Convection-enhanced delivery” refers to any non-manual delivery ofagents. In the context of the present invention, examples ofconvection-enhanced delivery (CED) of AAV can be achieved by infusionpumps or by osmotic pumps. A more detailed description of CED is foundbelow.

The terms “subject”, “individual” or “patient” are used interchangeablyherein and refer to a vertebrate, preferably a mammal. Mammals include,but are not limited to, murines, simians, humans, farm animals, sportanimals and pets.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages.

B. General Methods

The present invention is based on the surprising discovery that AAVvector-mediated GDNF gene delivery results in the durable andsubstantial expression of GDNF after a single intramuscular injection.As shown in the examples herein, substantial GDNF expression wasachieved in a large number of myofibers and reached as high as nanogramlevels in muscle and accumulated at the neuromuscular junctions.Expression persisted for at least 10 months. Moreover, the expressedGDNF was retrogradely transported through axons to corresponding spinalcord motoneurons. The transgene GDNF prevented degeneration ofmotoneurons, preserved the axons innervating the muscle, and inhibitedmuscle atrophy. Significantly, four-limb injection of AAV-GDNF in anamyotrophic lateral sclerosis (ALS) animal model postponed diseaseonset, delayed the progression of motor dysfunction, and prolonged thelife-span in treated animals. Consistent with these functional benefits,marked histopathologic amelioration such as reduced loss and atrophy ofspinal cord motoneurons, more frequent intact neuromuscular connections,and reduced myofiber atrophy were also observed. Accordingly, deliveryof GDNF via recombinant AAV virions provides a powerful and efficaciousmethod for treating motoneuron disease, such as ALS.

The method described herein provides for the direct, in vivo injectionof recombinant AAV virions into muscle tissue, preferably skeletalmuscle, e.g., by intramuscular injection, as well as for the in vitrotransduction of muscle cells which can subsequently be introduced into asubject for treatment. The methods described herein can be used to treata number of motoneuron diseases such as any of the various amyotrophiessuch as hereditary amyotrophies including hereditary spinal muscularatrophy, acute infantile spinal muscular atrophy such as Werdnig-Hoffmandisease, progressive muscular atrophy in children such as the proximal,distal type and bulbar types, spinal muscular atrophy of adolescent oradult onset including the proximal, scapuloperoneal, facioscapulohumeraland distal types, amyotrophic lateral sclerosis (ALS) and primarylateral sclerosis (PLS).

As explained above, GDNF is a protein that may be identified in orobtained from glial cells and that exhibits neurotrophic activity. GDNFis an approximately 39 kD glycosylated protein that exists as ahomodimer in its native form. GDNF is initially translated as apre-pro-GDNF polypeptide and proteolytic processing of the signalsequence and the “pro” portion of the molecule result in production of amature form of GDNF. In humans and rodents, a single gene gives rise toalternatively spliced forms. See, e.g., U.S. Pat. No. 6,362,319,incorporated herein by reference in its entirety. Both forms contain aconsensus signal peptide sequence and a consensus sequence forproteolytic processing. Proteolytic cleavage yields identical mature 134amino acid residue forms. Thus, GDNF polynucleotides for use in thepresent AAV vectors may encode either or both of these forms, may encodethe entire pre-pro-molecule, the pre-molecule, the pro-molecule, themature GDNF polypeptide, or biologically active variants of these forms,as defined above.

A number of GDNF polynucleotide and amino acid sequences are known.three representative mammalian GDNF sequences are depicted in FIGS. 7, 8and 9 herein. In particular, a human GDNF nucleotide and amino acidsequence is shown in FIG. 7 (SEQ ID NOS:1 and 2). A rat GDNF nucleotideand amino acid sequence is shown in FIG. 8 (SEQ ID NOS:3 and 4) and amouse GDNF nucleotide and amino acid sequence is shown in FIG. 9 (SEQ IDNOS:6 and 7). The degree of homology between the rat and human proteinsis about 93% and all mammalian GDNFs have a similarly high degree ofhomology. Additional GDNF nucleotide and amino acid sequences are knownin the art. See, e.g., U.S. Pat. Nos. 6,221,376 and 6,363,319,incorporated herein by reference in their entireties, and Lin et al.,Science (1993) 260:1130-1132 for rat and human sequences, as well asNCBI accession numbers AY052832, AJ001896, AF053748, AF063586 and L19063for human sequences; NCBI accession numbers AF184922, AF497634, X92495,NM019139 for rat sequences; NCBI accession number AF516767 for a giantpanda sequence; NCBI accession numbers XM122804, NM010275, D88351S1,D49921, U36449, U37459, U66195 for mouse sequences; NCBI accessionnumber AF469665 for a Nipponia nippon sequence; NCBI accession numberAF106678 for a Macaca mulatta sequence; and NCBI accession numbersNM131732 and AF329853 for zebrafish sequences. As explained above, anyof these sequences, as well as variants thereof, such as sequencessubstantially homologous and functionally equivalent to these sequences,will find use in the present methods.

The efficacy of AAV-delivered GDNF polynucleotides can be tested in anyof a number of animal models of the above diseases, known in the art.For example, scientifically accepted and widely used animal models forthe study of motoneuron disorders such as ALS are transgenic mice withan ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) gene (mSOD1G93Aand/or mSOD1G37R). These mice develop a dominantly inherited adult-onsetparalytic disorder with many of the clinical and pathological featuresof familial ALS. See, e.g., Gurney et al., Science (1994) 264:1772-1775;Nagano et al., Life Sci (2002) 72:541-548. Other animal models includetwo naturally occurring murine models (progressive motor neuronopathy(pmn) and wobbler). See, e.g., Haegggeli and Kato, Neurosci. Lett.(2002) 335:39-43, for descriptions of these mouse models. For a reviewof various animal models for use in studying motoneuron diseases such asALS, see, e.g., Jankowsky et al., Curr Neurol Neurosci. Rep. (2002)2:457-464; Elliott, J.L., Neurobiol. Dis. (1999) 6:310-20; and Borcheltet al., Brain Pathol. (1998) 8:735-757.

Additionally, several in vitro model systems are known which use cells,tissue culture and histological methods for studying motoneuron disease.For example, a rat spinal cord organotypic slice subjected to glutamateexcitotoxicity is useful as a model system to test the effectiveness ofneurotrophic factors in preventing motor neuron degeneration. Corse etal., Neurobiol. Dis. (1999) 6:335-346. For a discussion of in vitrosystems for use in studying ALS, see, e.g., Bar, P.R., Eur. J Pharmacol.(2000) 405:285-295; Silani et al., J Neurol. (2000) 247 Suppl 1:I28-36;Martin et al., Int. J Mol. Med. (2000) 5:3-13.

Animal models of other neurodegenerative diseases have been describedand are useful for evaluating the therapeutic efficacy of AAV-deliveredGDNF polynucleotides in the treatment of motoneuron disorders inaddition to ALS. See, for example, Katsuno et al., Neuron (2002)35:843-854 for a transgenic mouse model for evaluating spinal and bulbarmuscular atrophy (SBMA); Ford et al., Microb. Pathog. (2002) 33:97-107for a description of animal models for human paralytic poliomyelitis.

Recombinant AAV virions comprising GDNF coding sequences may be producedusing a variety of art-recognized techniques described more fully below.Wild-type AAV and helper viruses may be used to provide the necessaryreplicative functions for producing rAAV virions (see, e.g., U.S. Pat.No. 5,139,941, incorporated herein by reference in its entirety).Alternatively, a plasmid, containing helper function genes, incombination with infection by one of the well-known helper viruses canbe used as the source of replicative functions (see e.g., U.S. Pat. No.5,622,856 and U.S. Pat. No. 5,139,941, both incorporated herein byreference in their entireties). Similarly, a plasmid, containingaccessory function genes can be used in combination with infection bywild-type AAV, to provide the necessary replicative functions. Thesethree approaches, when used in combination with a rAAV vector, are eachsufficient to produce rAAV virions. Other approaches, well known in theart, can also be employed by the skilled artisan to produce rAAVvirions.

In a preferred embodiment of the present invention, a tripletransfection method (described in detail in U.S. Pat. No. 6,001,650,incorporated by reference herein in its entirety) is used to producerAAV virions because this method does not require the use of aninfectious helper virus, enabling rAAV virions to be produced withoutany detectable helper virus present. This is accomplished by use ofthree vectors for rAAV virion production: an AAV helper function vector,an accessory function vector, and a rAAV expression vector. One of skillin the art will appreciate, however, that the nucleic acid sequencesencoded by these vectors can be provided on two or more vectors invarious combinations.

As explained herein, the AAV helper function vector encodes the “AAVhelper function” sequences (i.e., rep and cap), which function in transfor productive AAV replication and encapsidation. Preferably, the AAVhelper function vector supports efficient AAV vector production withoutgenerating any detectable wt AAV virions (i.e., AAV virions containingfunctional rep and cap genes). An example of such a vector, pHLP19 isdescribed in U.S. Pat. No. 6,001,650, incorporated herein by referencein its entirety. The rep and cap genes of the AAV helper function vectorcan be derived from any of the known AAV serotypes, as explained above.For example, the AAV helper function vector may have a rep gene derivedfrom AAV-2 and a cap gene derived from AAV-6; one of skill in the artwill recognize that other rep and cap gene combinations are possible,the defining feature being the ability to support rAAV virionproduction.

The accessory function vector encodes nucleotide sequences for non-AAVderived viral and/or cellular functions upon which AAV is dependent forreplication (i.e., “accessory functions”). The accessory functionsinclude those functions required for AAV replication, including, withoutlimitation, those moieties involved in activation of AAV genetranscription, stage specific AAV mRNA splicing, AAV DNA replication,synthesis of cap expression products, and AAV capsid assembly.Viral-based accessory functions can be derived from any of thewell-known helper viruses such as adenovirus, herpesvirus (other thanherpes simplex virus type-1), and vaccinia virus. In a preferredembodiment, the accessory function plasmid pLadeno5 is used (detailsregarding pLadeno5 are described in U.S. Pat. No. 6,004,797,incorporated herein by reference in its entirety). This plasmid providesa complete set of adenovirus accessory functions for AAV vectorproduction, but lacks the components necessary to formreplication-competent adenovirus.

In order to further an understanding of the invention, a more detaileddiscussion is provided below regarding recombinant AAV expressionvectors, AAV helper and accessory functions, compositions comprising AAVvirions, as well as delivery of virions.

Recombinant AAV Expression Vectors

Recombinant AAV (rAAV) expression vectors are constructed using knowntechniques to at least provide as operatively linked components in thedirection of transcription, control elements including a transcriptionalinitiation region, the GDNF polynucleotide of interest and atranscriptional termination region. The control elements are selected tobe functional in a mammalian muscle cell. The resulting construct whichcontains the operatively linked components is bounded (5′ and 3′) withfunctional AAV ITR sequences.

The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin,R.M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. “Parvoviridae andtheir Replication” in Fundamental Virology, 2nd Edition, (B. N. Fieldsand D. M. Knipe, eds.) for the AAV-2 sequence. AAV ITRs used in thevectors of the invention need not have a wild-type nucleotide sequence,and may be altered, e.g., by the insertion, deletion or substitution ofnucleotides. Additionally, AAV ITRs may be derived from any of severalAAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAV-6, AAV-7 and AAV-8, etc. Furthermore, 5′ and 3′ ITRs whichflank a selected nucleotide sequence in an AAV expression vector neednot necessarily be identical or derived from the same AAV serotype orisolate, so long as they function as intended, i.e., to allow forexcision and rescue of the sequence of interest from a host cell genomeor vector, and to allow integration of the DNA molecule into therecipient cell genome when AAV Rep gene products are present in thecell.

Suitable GDNF polynucleotide molecules for use in AAV vectors will beless than about 5 kilobases (kb) in size. The selected polynucleotidesequence is operably linked to control elements that direct thetranscription or expression thereof in the subject in vivo. Such controlelements can comprise control sequences normally associated with theselected gene. Alternatively, heterologous control sequences can beemployed. Useful heterologous control sequences generally include thosederived from sequences encoding mammalian or viral genes. Examplesinclude, but are not limited to, neuron-specific enolase promoter, aGFAP promoter, the SV40 early promoter, mouse mammary tumor virus LTRpromoter; adenovirus major late promoter (Ad MLP); a herpes simplexvirus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMVimmediate early promoter region (CMVIE), a rous sarcoma virus (RSV)promoter, synthetic promoters, hybrid promoters, and the like. Inaddition, sequences derived from nonviral genes, such as the murinemetallothionein gene, will also find use herein. Such promoter sequencesare commercially available from, e.g., Stratagene (San Diego, Calif.).

For purposes of the present invention, muscle-specific and induciblepromoters, enhancers and the like, will be of particular use. Suchcontrol elements include, but are not limited to, those derived from theactin and myosin gene families, such as from the myoD gene family(Weintraub et al. (1991) Science 251:761-766); the myocyte-specificenhancer binding factor MEF-2 (Cserjesi and Olson (1991) Mol. Cell Biol.11:4854-4862); control elements derived from the human skeletal actingene (Muscat et al. (1987) Mol. Cell Biol. 7:4089-4099) and the cardiacactin gene; muscle creatine kinase sequence elements (Johnson et al.(1989) Mol. Cell Biol. 9:3393-3399) and the murine creatine kinaseenhancer (mCK) element; control elements derived from the skeletalfast-twitch troponin C gene, the slow-twitch cardiac troponin C gene andthe slow-twitch troponin I gene; hypoxia-inducible nuclear factors(Semenza et al. (1991) Proc. Natl. Acad. Sci. USA 88:5680-5684; Semenzaet al. J Biol. Chem. 269:23757-23763); steroid-inducible elements andpromoters, such as the glucocorticoid response element (GRE) (Mader andWhite (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607); the fusionconsensus element for RU486 induction; elements that provide fortetracycline regulated gene expression (Dhawan et al. (1995) Somat.Cell. Mol. Genet. 21:233-240; Shockett et al. (1995) Proc. Natl. Acad.Sci. USA 92:6522-6526; and inducible, synthetic humanized promoters(Rivera et al. (1996) Nature Med. 2:1028-1032). These and otherregulatory elements can be tested for potential in vivo efficacy usingthe in vitro myoblast model, which mimics quiescent in vivo musclephysiology.

The AAV expression vector which harbors the GDNF polynucleotide moleculeof interest bounded by AAV ITRs, can be constructed by directlyinserting the selected sequence(s) into an AAV genome which has had themajor AAV open reading frames (“ORFs”) excised therefrom. Other portionsof the AAV genome can also be deleted, so long as a sufficient portionof the ITRs remain to allow for replication and packaging functions.Such constructs can be designed using techniques well known in the art.See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; InternationalPublication Nos. WO 92/01070 (published 23 Jan. 1992) and WO 93/03769(published 4 Mar. 1993); Lebkowski et al. (1988) Molec. Cell. Biol.8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring HarborLaboratory Press); Carter (1992) Current Opinion in Biotechnology3:533-539; Muzyczka (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin (1994) Human Gene Therapy 5:793-801; Shelling andSmith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med.179:1867-1875.

Alternatively, AAV ITRs can be excised from the viral genome or from anAAV vector containing the same and fused 5′ and 3′ of a selected nucleicacid construct that is present in another vector using standard ligationtechniques, such as those described in Sambrook et al., supra. Forexample, ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10 mMMgCl₂, 10 mM DTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 40 μM ATP,0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for “sticky end”ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C.(for “blunt end” ligation). Intermolecular “sticky end” ligations areusually performed at 30-100 μg/ml total DNA concentrations (5-100 nMtotal end concentration). AAV vectors which contain ITRs have beendescribed in, e.g., U.S. Pat. No. 5,139,941. In particular, several AAVvectors are described therein which are available from the American TypeCulture Collection (“ATCC”) under Accession Numbers 53222, 53223, 53224,53225 and 53226.

Additionally, chimeric genes can be produced synthetically to includeAAV ITR sequences arranged 5′ and 3′ of one or more selected nucleicacid sequences. Preferred codons for expression of the chimeric genesequence in mammalian muscle cells can be used. The complete chimericsequence is assembled from overlapping oligonucleotides prepared bystandard methods. See, e.g., Edge (1981) Nature 292:756; Nambair et al.(1984) Science 223:1299; Jay et al. (1984) J Biol. Chem. (1984)259:6311.

For the purposes of the invention, suitable host cells for producingrAAV virions from the AAV expression vectors include microorganisms,yeast cells, insect cells, and mammalian cells, that can be, or havebeen, used as recipients of a heterologous DNA molecule and that arecapable of growth in suspension culture. The term includes the progenyof the original cell which has been transfected. Thus, a “host cell” asused herein generally refers to a cell which has been transfected withan exogenous DNA sequence. Cells from the stable human cell line, 293(readily available through, e.g., the American Type Culture Collectionunder Accession Number ATCC CRL1573) are preferred in the practice ofthe present invention. Particularly, the human cell line 293 is a humanembryonic kidney cell line that has been transformed with adenovirustype-5 DNA fragments (Graham et al. (1977) J Gen. Virol. 36:59), andexpresses the adenoviral E1a and E1b genes (Aiello et al. (1979)Virology 94:460). The 293 cell line is readily transfected, and providesa particularly convenient platform in which to produce rAAV virions.

AAV Helper Functions

Host cells containing the above-described AAV expression vectors must berendered capable of providing AAV helper functions in order to replicateand encapsidate the nucleotide sequences flanked by the AAV ITRs toproduce rAAV virions. AAV helper functions are generally AAV-derivedcoding sequences which can be expressed to provide AAV gene productsthat, in turn, function in trans for productive AAV replication. AAVhelper functions are used herein to complement necessary AAV functionsthat are missing from the AAV expression vectors. Thus, AAV helperfunctions include one, or both of the major AAV ORFs, namely the rep andcap coding regions, or functional homologues thereof

By “AAV rep coding region” is meant the art-recognized region of the AAVgenome which encodes the replication proteins Rep 78, Rep 68, Rep 52 andRep 40. These Rep expression products have been shown to possess manyfunctions, including recognition, binding and nicking of the AAV originof DNA replication, DNA helicase activity and modulation oftranscription from AAV (or other heterologous) promoters. The Repexpression products are collectively required for replicating the AAVgenome. For a description of the AAV rep coding region, see, e.g.,Muzyczka, N. (1992) Current Topics in MicrobioL and Immunol. 158:97-129;and Kotin, R.M. (1994) Human Gene Therapy 5:793-801. Suitable homologuesof the AAV rep coding region include the human herpesvirus 6 (HHV-6) repgene which is also known to mediate AAV-2 DNA replication (Thomson etal. (1994) Virology 204:304-311).

By “AAV cap coding region” is meant the art-recognized region of the AAVgenome which encodes the capsid proteins VP1, VP2, and VP3, orfunctional homologues thereof. These Cap expression products supply thepackaging functions which are collectively required for packaging theviral genome. For a description of the AAV cap coding region, see, e.g.,Muzyczka, N. and Kotin, R. M. (supra).

AAV helper functions are introduced into the host cell by transfectingthe host cell with an AAV helper construct either prior to, orconcurrently with, the transfection of the AAV expression vector. AAVhelper constructs are thus used to provide at least transient expressionof AAV rep and/or cap genes to complement missing AAV functions that arenecessary for productive AAV infection. AAV helper constructs lack AAVITRs and can neither replicate nor package themselves.

These constructs can be in the form of a plasmid, phage, transposon,cosmid, virus, or virion. A number of AAV helper constructs have beendescribed, such as the commonly used plasmids pAAV/Ad and pIM29+45 whichencode both Rep and Cap expression products. See, e.g., Samulski et al.(1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol.65:2936-2945. A number of other vectors have been described which encodeRep and/or Cap expression products. See, e.g., U.S. Pat. No. 5,139,941.

Both AAV expression vectors and AAV helper constructs can be constructedto contain one or more optional selectable markers. Suitable markersinclude genes which confer antibiotic resistance or sensitivity to,impart color to, or change the antigenic characteristics of those cellswhich have been transfected with a nucleic acid construct containing theselectable marker when the cells are grown in an appropriate selectivemedium. Several selectable marker genes that are useful in the practiceof the invention include the hygromycin B resistance gene (encodingAminoglycoside phosphotranferase (APH)) that allows selection inmammalian cells by conferring resistance to G418 (available from Sigma,St. Louis, Mo.). Other suitable markers are known to those of skill inthe art.

AAV Accessory Functions

The host cell (or packaging cell) must also be rendered capable ofproviding non AAV-derived functions, or “accessory functions,” in orderto produce rAAV virions. Accessory functions are nonAAV-derived viraland/or cellular functions upon which AAV is dependent for itsreplication. Thus, accessory functions include at least those nonAAVproteins and RNAs that are required in AAV replication, including thoseinvolved in activation of AAV gene transcription, stage specific AAVMRNA splicing, AAV DNA replication, synthesis of Cap expression productsand AAV capsid assembly. Viral-based accessory functions can be derivedfrom any of the known helper viruses.

In particular, accessory functions can be introduced into and thenexpressed in host cells using methods known to those of skill in theart. Typically, accessory functions are provided by infection of thehost cells with an unrelated helper virus. A number of suitable helperviruses are known, including adenoviruses; herpesviruses such as herpessimplex virus types 1 and 2; and vaccinia viruses. Nonviral accessoryfunctions will also find use herein, such as those provided by cellsynchronization using any of various known agents. See, e.g., Buller etal. (1981) J. Virol. 40:241-247; McPherson et al. (1985) Virology147:217-222; Schlehofer et al. (1986) Virology 152:110-117.

Alternatively, accessory functions can be provided using an accessoryfunction vector as defined above. See, e.g., U.S. Pat. No. 6,004,797 andInternational Publication No. WO 01/83797, incorporated herein byreference in its entirety. Nucleic acid sequences providing theaccessory functions can be obtained from natural sources, such as fromthe genome of an adenovirus particle, or constructed using recombinantor synthetic methods known in the art. As explained above, it has beendemonstrated that the full-complement of adenovirus genes are notrequired for accessory helper functions. In particular, adenovirusmutants incapable of DNA replication and late gene synthesis have beenshown to be permissive for AAV replication. Ito et al., (1970) J Gen.Virol. 9:243; Ishibashi et al, (1971) Virology 45:317. Similarly,mutants within the E2B and E3 regions have been shown to support AAVreplication, indicating that the E2B and E3 regions are probably notinvolved in providing accessory functions. Carter et al., (1983)Virology 126:505. However, adenoviruses defective in the E1 region, orhaving a deleted E4 region, are unable to support AAV replication. Thus,E1A and E4 regions are likely required for AAV replication, eitherdirectly or indirectly. Laughlin et al., (1982) J Virol. 41:868; Janiket al., (1981) Proc. Natl. Acad. Sci. USA 78:1925; Carter et al., (1983)Virology 126:505. Other characterized Ad mutants include: E1B (Laughlinet al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., (1980)Virology 104:502); E2A (Handa et al., (1975) J Gen. Virol. 29:239;Strauss et al., (1976) J. Virol. 17:140; Myers et al., (1980) J. Virol.35:665; Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:2927; Myers etal., (1981) J. Biol. Chem. 256:567); E2B (Carter, Adeno-Associated VirusHelper Functions, in I CRC Handbook ofParvoviruses (P. Tijssen ed.,1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al.(1983),supra; Carter (1995)). Although studies of the accessory functionsprovided by adenoviruses having mutations in the E1B coding region haveproduced conflicting results, Samulski et al., (1988) J. Virol.62:206-210, recently reported that E1B55k is required for AAV virionproduction, while E1B19k is not. In addition, International PublicationWO 97/17458 and Matshushita et al., (1998) Gene Therapy 5:938-945,describe accessory function vectors encoding various Ad genes.

Particularly preferred accessory function vectors comprise an adenovirusVA RNA coding region, an adenovirus E4 ORF6 coding region, an adenovirusE2A 72 kD coding region, an adenovirus E1A coding region, and anadenovirus E1B region lacking an intact E1B55k coding region. Suchvectors are described in International Publication No. WO 01/83797.

As a consequence of the infection of the host cell with a helper virus,or transfection of the host cell with an accessory function vector,accessory functions are expressed which transactivate the AAV helperconstruct to produce AAV Rep and/or Cap proteins. The Rep expressionproducts excise the recombinant DNA (including the DNA of interest) fromthe AAV expression vector. The Rep proteins also serve to duplicate theAAV genome. The expressed Cap proteins assemble into capsids, and therecombinant AAV genome is packaged into the capsids. Thus, productiveAAV replication ensues, and the DNA is packaged into rAAV virions.

Following recombinant AAV replication, rAAV virions can be purified fromthe host cell using a variety of conventional purification methods, suchas column chromatography, CsCl gradients, and the like. For example, aplurality of column purification steps can be used, such as purificationover an anion exchange column, an affinity column and/or a cationexchange column. See, for example, International Publication No. WO02/12455. Further, if infection is employed to express the accessoryfunctions, residual helper virus can be inactivated, using knownmethods. For example, adenovirus can be inactivated by heating totemperatures of approximately 60° C. for, e.g., 20 minutes or more. Thistreatment effectively inactivates only the helper virus since AAV isextremely heat stable while the helper adenovirus is heat labile.

The resulting rAAV virions containing the GDNF nucleotide sequence ofinterest can then be used for gene delivery using the techniquesdescribed below.

Compositions

Compositions will comprise sufficient genetic material to produce atherapeutically effective amount of the GDNF of interest, i.e., anamount sufficient to reduce or ameliorate symptoms of the disease statein question or an amount sufficient to confer the desired benefit. Thecompositions will also contain a pharmaceutically acceptable excipient.Such excipients include any pharmaceutical agent that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition, and which may be administered without undue toxicity.Pharmaceutically acceptable excipients include, but are not limited to,sorbitol, any of the various TWEEN compounds, and liquids such as water,saline, glycerol and ethanol. Pharmaceutically acceptable salts can beincluded therein, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Additionally, auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles. A thorough discussion ofpharmaceutically acceptable excipients is available in REMINGTON'SPHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

One particularly useful formulation comprises recombinant AAV virions incombination with one or more dihydric or polyhydric alcohols, and,optionally, a detergent, such as a sorbitan ester. See, for example,International Publication No. WO 00/32233.

As is apparent to those skilled in the art in view of the teachings ofthis specification, an effective amount of viral vector which must beadded can be empirically determined. Representative doses are detailedbelow. Administration can be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosages of administration arewell known to those of skill in the art and will vary with the viralvector, the composition of the therapy, the target cells, and thesubject being treated. Single and multiple administrations can becarried out with the dose level and pattern being selected by thetreating physician.

It should be understood that more than one transgene can be expressed bythe delivered recombinant virion. Alternatively, separate vectors, eachexpressing one or more different transgenes, can also be delivered asdescribed herein. Furthermore, it is also intended that the viralvectors delivered by the methods of the present invention be combinedwith other suitable compositions and therapies. Where the transgene isunder the control of an inducible promoter, certain systemicallydelivered compounds such as muristerone, ponasteron, tetracyline oraufin may be administered in order to regulate expression of thetransgene.

Delivery of AAV Virions

Recombinant AAV virions may be introduced into muscle cells using eitherin vivo or in vitro (also termed ex vivo) transduction techniques. Iftransduced in vitro, the desired recipient cell, preferably a skeletalmuscle cell, will be removed from the subject, transduced with rAAVvirions and reintroduced into the subject. Alternatively, syngeneic orxenogeneic cells can be used where those cells will not generate aninappropriate immune response in the subject.

Suitable methods for the delivery and introduction of transduced cellsinto a subject have been described. For example, cells can be transducedin vitro by combining recombinant AAV virions with cells to betransduced in appropriate media, and those cells harboring the DNA ofinterest can be screened using conventional techniques such as Southernblots and/or PCR, or by using selectable markers. Transduced cells canthen be formulated into pharmaceutical compositions, as described above,and the composition introduced into the subject by various techniques asdescribed below, in one or more doses.

Recombinant AAV virions or cells transduced in vitro may be delivereddirectly to muscle by injection with a needle, catheter or relateddevice, using techniques known in the art. For in vivo delivery, therAAV virions will be formulated into pharmaceutical compositions and oneor more dosages may be administered directly in the indicated manner. Atherapeutically effective dose will include on the order of from about10⁸/kg to 10¹⁶/kg of the rAAV virions, more preferably 10¹⁰/kg to10¹⁴/kg, and even more preferably about 10¹¹/kg to 10¹³/kg of the rAAVvirions (or viral genomes, also termed “vg”), or any value within theseranges.

One mode of administration of recombinant AAV virions uses aconvection-enhanced delivery (CED) system. In this way, recombinantvirions can be delivered to many cells over large areas of muscle.Moreover, the delivered vectors efficiently express transgenes in musclecells. Any convection-enhanced delivery device may be appropriate fordelivery of viral vectors. In a preferred embodiment, the device is anosmotic pump or an infusion pump. Both osmotic and infusion pumps arecommercially available from a variety of suppliers, for example AlzetCorporation, Hamilton Corporation, Alza, Inc., Palo Alto, Calif.).Typically, a viral vector is delivered via CED devices as follows. Acatheter, cannula or other injection device is inserted into appropriatemuscle tissue in the chosen subject, such as skeletal muscle. For adetailed description regarding CED delivery, see U.S. Pat. No.6,309,634, incorporated herein by reference in its entirety.

Other modes of administration that will find particular use with musclesuse histamine or isolated limb perfusion (a technique where the vascularsupply to a limb is isolated from systemic circulation before infusionof the composition in question) for increasing vector spread in themuscle, all well known techniques in the art. See, e.g., Schaadt et al.,J. Extra Corpor. Technol. (2002) 34:130-143; Lejeune et al., Surg.Oncol. Clin. N. Am. (2001) 10:821-832; Fraser et al., AORN J. (1999)70:642-647, 649, 651-653.

C. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

EXAMPLE 1 Construction of Recombinant AAV Vectors

In order to distinguish AAV-delivered GDNF from its endogenouscounterpart, an AAV vector was constructed encoding a recombinant fusionGDNF protein, tagged with the FLAG peptide (AAV-GDNF-FLAG), forrecognition by a specific antibody to the FLAG epitope. Briefly, AAVvector plasmid pAAV-GDNF-FLAG was derived from a previously describedpAAV-GDNF plasmid (Fan et al., Neurosci. Lett. (1998) 248:61-64). Thisplasmid contains the mouse GDNF cDNA (Matsushita et al., Gene (1997)203:149-157) tagged by the FLAG sequence (DYKDDDDK (SEQ ID NO:5) at thecarboxyl terminus under the human cytomegalovirus (CMV) immediate-earlypromoter, with human growth hormone first intron and simian virus 40(SV40) polyadenylation signal sequence between the inverted terminalrepeats (ITR) of the AAV-2 genome.

AAV vector plasmid pAAV-LacZ, auxiliary plasmid pHLP19 and pladenol havepreviously been described (Fan et al., Neurosci. Lett. (1998) 248:61-64;Shen et al., Hum. Gene Ther. (2000) 11:1509-1519). Pladeno5 is describedin U.S. Pat. No. 6,004,797. Subconfluent human 293 cells weretransiently transfected with vector plasmid and helper plasmid using thecalcium phosphate co-precipitation method. Seventy-two hours aftertransfection, cells were harvested and lysed by freeze and thaw cycles.AAV vectors (AAV-GDNF-FLAG and AAV-LacZ) were purified using twosequential continuous CsCl gradients, as described previously(Matsushita, et al, Gene Therapy (1998) 5:938-945). The final particletiter of the AAV-GDNF-FLAG was 1.6×10¹³ vector genome copies/ml andAAV-LacZ was 2.1×10¹³ vector genome copies/ml, as estimated byquantitative DNA dot-blot hybridization analysis.

EXAMPLE 2 In Vitro Expression of AAV-GDNF-FLAG

To detect the in vitro expression of GDNF-FLAG fusion protein, humanembryonic kidney (HEK) 293 cells (readily available through, e.g., theAmerican Type Culture Collection under Accession Number ATCC CRL1573)were seeded on 35 mm-diameter dishes and allowed to proliferateovernight in complete medium. Cells were transduced with AAV-GDNF-FLAGor AAV-LacZ (0.5-18×10³ vector genome copies/cell) in medium suppliedwith 2% fetal bovine serum. 48 hours after transduction, conditionedmedium (CM) and cell lysates were collected.

GDNF levels in CM and 293 cell lysate were measured by ELISA (GDNFE_(max) ImmunoAssay System, Promega). For the detection in cell lysate,cells were homogenized in lysis buffer (137×10⁻³ mol/L NaCl, 20×10⁻³mol/L Tris [pH 8.0], 1% NP40, 10% glycerol, supplied by ProteaseInhibitor Cocktail Tablets Complete Mini [Roche]), ultrasonicated andcentrifuged at 4° C. The supernatants were acidified and thenneutralized to pH 7.4 before assay. Acidification has been reported toenhance detection of neurotrophic factors (Okragly et al., Exp. Neurol.(1997) 145:592-596). Triplicate samples were processed in 96-well platesaccording to the detailed protocol provided by Promega. Briefly, plateswere coated with anti-GDNF monoclonal antibody, blocked and incubatedwith GDNF standards or samples. Plates were then incubated sequentiallywith chicken anti-human GDNF polyclonal antibody, anti-chicken1gY-peroxidase conjugate, followed by peroxidase substrate andtetramethylbenzidine solution for color development. The reaction wasstopped with 1N HCl and the absorbance was read at 450 nm. Levels ofGDNF were expressed as pg/mg tissue. The assay sensitivity ranged from16 pg/ml to 1,000 pg/ml.

ELISA analysis showed high levels of GDNF expression and secretion inAAV-GDNF-FLAG-transduced cells (FIGS. 1A and 1B). As expected, GDNFlevels in the CM were much higher than the cell lysate, indicating thatthe GDNF could be secreted by cells. Moreover, at the range detected,the amount of GDNF in CM and cell lysates showed a vector genome copiesper cell/dose-dependent increase. In the non-transduced orAAV-LacZ-transduced 293 cells, GDNF levels were much lower, barely atthe detection limit of the ELISA analysis.

EXAMPLE 3 In Vivo Expression of GDNF in Injected Gastrocnemius Muscles

Male C57BL/6J mice (7 weeks old) were injected with either AAV-GDNF-FLAG(n=32) or AAV-LacZ (n=21) virions in the left hindlimb gastrocnemiusmuscles (2×10¹⁰ viral genome copies in 24 μl PBS/3 sites) percutaneouslyusing a microsyringe connected to a 27-gauge needle. As a sham control,the right gastrocnemius muscle was injected with same volume of PBS. Nomorbidity or morality was observed in nice during the experimentalperiod. At the indicated time (see FIG. 2), gastrocnemius muscles weredissected, rapidly frozen in liquid nitrogen-cooled isopentane andstored at −80° C. for ELISA analysis or Cryostat sectioning. Mice werethen perfused with ice-cold PBS followed by 4% paraphormaldehyde (PFA).Spinal cord was dissected, post-fixed for 4 hours in 4% PFA andcryoprotected by soaking sequentially in 10%, 20% and 30% sucrose at 4°C. overnight. Serial transverse Cryostat sections of frozen muscletissue (10 μm) were thawed mounted in slides, coated with gelatin, andcompletely dried before storing at −80° C. Serial transverse sections oflumbar spinal cord were cut on freezing microtome at 30 μm thickness,and stored in PBS at 4° C.

GDNF ELISAs were performed as described above. For β-galactosidase(β-Gal) histochemistry, muscle sections were fixed and stained for 4-6hours with β-Gal staining solution (500 μg/ml X-Gal, 5 mM potassiumhexacycanoferrate (III), 5 mM potassium hexacycanoferrate (II), and 2 mMmagnesium chloride in PBS) at 37° C. Spinal cord samples were stained asfree-floating sections and mounted in gelatin-coated slices and dried.Sections were counterstained with eosin for detection.Immunohistochemistry staining with anti-FLAG antibody was performed ongastrocnemius muscle cryostat sections for the purpose of distinguishingtransgene GDNF from endogenous GDNF. Muscle sections were fixed andtreated with 0.3% H₂O₂, then sequentially incubated with anti-FLAGantibody (1:1000, rabbit polyclonal anti-FLAG antibody, Sigma) overnightat 4° C. and a biotinylated secondary antibody to rabbit IgG (1:400) fortwo hours, and visualized using the avidin-biotinylated peroxidasecomplex procedure (Vectastain ABC lots. Vector Laboratories Inc.Burlingame, Calif.), using 3,3-diaminobenzidine (DAB) as a chromogen.For double immunofluorescence staining, muscle and spinal cord sectionswere stained with the Mouse-on-Mouse kit (M.O.M kit) (VectorLaboratories, Burlingame, Calif.), according to the manufacturer'sprotocol. Primary antibodies used for muscle sections were mouseanti-FLAG M₂ antibody (1:500, Sigma) and rabbit anti-GDNF antibody(1:1000, Santa Cruz). For spinal cord sections antibodies were rabbitanti-FLAG antibody (1:1000, Sigma) and mouse anti-NeuN antibody (1:200,Chemicon). Sections were then incubated with rhodamine orFITC-conjugated corresponding secondary antibodies for detection. Fordouble immunofluorescent staining of muscle sections with anti-FLAGantibody and α-bungarotoxin, sections were incubated with rabbitanti-FLAG antibody first, followed by incubation with FITC-conjugatedanti-rabbit secondary antibody and tetramethyl-rhodamine conjugatedα-bungarotoxin molecular probe (1:400, Molecular Probes, Inc., Eugene,USA). α-Bungarotoxin is a peptide extracted from Bungarus multicinctus,which specifically binds with high affinity to the α-subunit of thenicotinic AchR at the postsynaptic membrane of neuromuscular junctions.Immunofluorescent stained sections were viewed and photographs werecaptured with a confocal laser scanning microscope (TCS NT; Leica,Heidelberg, Germany).

ELISA Detection of GDNF levels in AAV-GDNF-FLAG-injected gastrocnemiusmuscles showed that GDNF expression could be detected 14 dayspostinjection, gradually increased over the first 2 months and remainedstable without significant diminution over 6 months, then showed slightreduction from 8 months postinjection (FIG. 2). The substantialexpression persisted at least 10 months, the last time point tested. Thegastrocnemius muscles injected with AAV-LacZ vector or PBS exhibitedvery low levels of GDNF.

In gastrocnemius muscles injected with AAV-GDNF-FLAG, substantial andsustained immunoreactive signals for FLAG were detected in a largenumber of myofibers, from 2 weeks to 10 months postinjection.Double-immunofluorescent staining with anti-GDNF and anti-FLAGantibodies showed co-localization of GDNF and FLAG immunoreactivity,confirming the presence of a GDNF-FLAG fusion protein. Intenseimmunoreactivity was concentrated in the vicinity of the sarcolemmas,suggesting its effective secretion by myofibers in vivo. In AAV-LacZ- orPBS-injected muscles, only very weak signals of GDNF could be detectedin the vicinity of sarcolemmas, while no immunosignals of FLAG wereseen. This is in agreement with ELISA results and indicated that theGDNF detected by ELISA in AAV-LacZ- or PBS-injected muscles wasendogenous GDNF.

To summarize, substantial transgene GDNF was expressed in vivo andexpression reached as high as nanogram levels from each gastrocnemiusmuscle and persisted for as long as at least 10 months. A slightdiminution was observed beginring at 8 months postinjection and isconsistent with published results of other groups (Verma and Somia,Nature (1997) 389:239-242) as well as our unpublished results in thestriatum. Without being bound by a particular theory, this diminution inexpression may be due to promoter shut-off, degradation of the vectorgenome, and/or turnover of the transduced myofibers (Verma and Somia,Nature (1997) 389:239-242; Rabinowitz and Samulski, Curr. Opin.Biotechnol. (1998) 9:470-475).

According to the neurotrophic theory, neurites connect with theirtargets to gain access to target-derived neurotrophic factors for neuronsurvival. In particular, neurotrophic factor is synthesized and releasedby the targets of neurotrophic factor-dependent axons, where it is boundby receptors on axon terminals, taken up, and retrogradely transportedto the cell body (DeStefano, P.S., Exp. Neurol. (1993) 124:56-69). Todetermine whether transgene-delivered GDNF-FLAG also followed theneurotrophic theory of GDNF, double-immunofluorescent staining withanti-FLAG antibody and rhodamine-conjugated α-bungarotoxin was performedon gastrocnemius muscle sections. Confocal microscopy showedco-localization of more intense immunoreactivity for FLAG with signalsof α-bungarotoxin, indicating the concentration of transgene GDNF-FLAGfusion protein to regions of neuromuscular junctions (NMJs). Theaccumulation of transgene GDNF at NMJs suggested that, consistent withthe neurotrophic theory, NMJ is a site for transgene GDNF uptake bynerve terminals as a target-derived neurotrophic factor. In AAV-LacZ- orPBS-injected sections, only rhodamine signals indicating the NMJs couldbe detected, with no FLAG immunoreactive signal. In the gastrocnemiusmuscles injected with AAV-LacZ vector, β-gal activity was detected in alarge number of the myofibers but in an expression pattern totallydifferent than the transgene GDNF. The expression lasted from 2 weeks to10 months postinjection. These results show that a single injection ofAAV-GDNF-FLAG in the gastrocnemius muscle is able to mediate durable andsubstantial expression of transgene GDNF, which was distributed mainlyin the vicinity of the sarcolemmas and accumulated at the NMJs.

The distribution of transgene GDNF-FLAG fusion protein in muscledetected by anti-FLAG antibody is similar to that of endogenous GDNFobserved in normal human muscles (Suzuki et al., J Comp. Neurol. (1998)402:303-312).

As a target-derived neurotrophic factor, endogenous GDNF produced byskeletal muscle functions via retrograde axonal transport from thetarget muscle tissue to motoneuronal cell bodies in the spinal cord(Mitsumoto, H., Muscle Nerve (1999) 22:1000-1021; Leitner et al., J.Neurosci. (1999) 19:9322-9331). Receptor-mediated retrograde transportof GDNF has been described in motoneurons of rats (Leitner et al., J.Neurosci. (1999) 19:9322-9331). Although the physiological significanceof retrograde transport is not completely understood, it is thought tobe of critical importance in axon-target communication and neuronalviability, probably reflecting the conveyance of a signal transductioncomplex (Neet and Capenot, Cell. Mol. Life Sci. (2001) 58:1221 -1235).

In order to determine whether transgene GDNF-FLAG fusion protein couldalso be retrogradely transported from muscle to spinal cord motoneurons,double-immunostaining with anti-FLAG and anti-NeuN (a specific marker ofneuron) antibodies, was performed on lumber 4 to 6 spinal cord sectionscorresponding with the innervation of gastrocnemius muscles. FLAGimmunoreactivity was detected in large size NeuN-positive cells ofventral horn ipsilateral to the AAV-GDNF-FLAG injected side. Their largesize (with diameters>20 μm), ventral horn distribution and NeuN-positivecharacteristics suggested that these FLAG immunoreactive cells wereα-motoneurons. As anti-FLAG antibody was used which excluded theinterference from endogenous GDNF, this evidenced the existence oftransgene GDNF-FLAG fusion protein in the motoneurons. In contrast, noFLAG staining was detected in the ventral horn of the contralateral sideas well as in both ventral horns of AAV-LacZ- or PBS-injected mice. Noβ-galactosidase signal was detected in the corresponding ventral hornsof spinal cord sections from AAV-LacZ-injected mice, in spite of thewide distribution β-galactosidase signals in the muscles.

Thus, transgene GDNF was successfully detected in the ipsilateralventral horn motoneurons of the spinal cord, following its detection ingastrocnemius muscle after AAV-GDNF-FLAG injection. The transgene GDNFmight have been delivered to the spinal cord via three differentavenues: systemic delivery, retrograde transport of AAV vectors orretrograde transport of the fusion protein itself. The restricteddistribution and ipsilateral presentation of transgenic GDNF inmotoneurons as well as its inability to pass through the blood-brainbarrier excludes the possibility of systematic delivery to the spinalcord. Additionally, β-galactosidase signal was not detected in spinalcord motoneurons of AAV-LacZ injected mice, indicating that the AAVvector itself was not delivered by retrograde transport. This evidencesthat the transgene GDNF-FLAG fusion protein detected in the spinal cordwas derived from the retrograde axonal transport of this protein frommuscles to the motoneurons, but not the AAV vector per se. This isconsistent with previous reports (Kordower et al., Science (2000)290:767-773;Wang et al., Gene Ther. (2002) 9:381-389). Retrograde axonaltransport of adenovirus vectors or lentiviral vectors has been reported(Ghadge et al., Gene Ther. (1995) 2:132-137; Haase et al., Nat. Med.(1997) 3:429-436; Desmaris et al., Mol. Ther. (2001) 4:149-156), butuntil the present discovery, no positive evidence supporting the massiveretrograde axonal transport of AAV-delivered gene products has ever beenreported.

In conclusion, the above examples demonstrate that AAV-GDNF-FLAGinjection to the gastrocnemius muscle supplies a continuous source oftransgene GDNF at the nerve terminals and constitutes a safe, durableand specific method to deliver GDNF to the motoneuronal bodies in thespinal cord. This, taken together with previous results that thetransgenic GDNF-FLAG fusion protein retains intact bioactivity as GDNF,shows the utility of AAV-mediated gene therapy for the treatment ofmotor neuron disease.

EXAMPLE 4 GDNF Transgene Expression in Muscles of ALS Mice

In order to determine whether AAV-mediated delivery of GDNF would beuseful for treating a motoneuron disease, such as ALS, the followingstudy was conducted.

A. Materials and Methods

Administration of Recombinant AAV virions.

Male transgenic mice with the G93A human SOD1 mutation (SOD1G93A) wereobtained from The Jackson Laboratory (Bar Harbor, Me.). pAAV-GDNF-FLAG,pAAV-LacZ, auxiliary plasmid pHLP19 and pladenol were as describedabove. AAV vectors were produced in human embryonic kidney (HEK) 293cells by triple transfection of vector plasmid and helper plasmidslisted above as described previously (Wang et al., Gene Ther. (2002)9:381-389). In brief, subconfluent 293 cells were transientlytransfected using the calcium phosphate method. 72 hours aftertransfection, the cells were collected and subjected to three cycles offreeze-thaw lysis (alternating between dry-ice-ethanol and 37° C.baths). AAV vectors were purified by two sequential continuous cesiumchloride density gradients and estimated for final particle titer byquantitative DNA dot-blot hybridization.

Before administration, AAV vectors were diluted in PBS to 1×10¹¹ genomecopies/100 μl. At 9 weeks of age, ALS mice were randomly assigned to onetreatment group that was injected with AAV-GDNF vector (n=12) or one oftwo control groups that were injected with AAV-LacZ vector (n=6) and thevehicle (n=5), respectively, into four limbs (gastrocnemius and tricepsbrachii muscles). The dosage was 30 μl for gastrocnemius and 20 μl fortriceps brachii muscles. Because mice injected with AAV-LacZ vector andthe vehicle were indistinguishable with regard to all variables testedduring the experimental period, the two groups were considered as onecontrol group for analysis. In another subgroup (n=7), all of the micehad AAV-GDNF vector injected into the muscles of the left forelimbs andhindlimbs and AAV-LacZ vector into those of the right ones.

Behavioral testing and mortality.

Mice were first given three days to become acquainted with the rotarodapparatus (Rota-Rod/7650; Ugo Basile, Comerio, Italy) before the test.For detection, mice were placed on the rotating rod at the speeds of 5,10, and 20 rpm, and the time each mouse remained on the rod wasregistered automatically. The onset of disease was defined as the timewhen the mouse could not remain on the rotarod for 7 min at a speed of20 rpm, as described previously (Li et al., Science (2000) 288:335-339).If the mouse remained on the rod for >7 min, the test was completed andscored as 7 min. Mice were tested every two days until they could nolonger perform the task. Mortality was scored as the age of death whenthe mouse was unable to right itself within 30 sec when placed on itsback in a supine position (Li et al., Science (2000) 288:335-339).

Tissue preparation.

One week before being sacrificed, mice were bilaterally injected withneural tracer cholera toxin subunit B (CTB) (0.1% in distilled H₂O, 3μl; List Biologic, Campbell, Calif.) into gastrocnemius muscles toselectively label motoneurons that retained axons innervating thetreated muscles. At the indicated times, gastrocnemius muscles weredissected out, weighed, rapidly frozen in liquid nitrogen-cooledisopentane, and then stored at −80° C. for immunohistochemistry or GDNFELISA analysis. After dissecting out the muscles, the mice were perfusedwith ice-cold PBS, followed by 4% paraformaldehyde (PFA). The spinalcord was dissected out, postfixed for 4 hr in 4% PFA, and thencryoprotected sequentially in sucrose.

GDNF ELISA.

To determine muscle GDNF levels, tissues were homogenized at a w/v ratioof 100 mg/ml in lysis buffer (137×10⁻³ mol/1 NaCl, 20×10⁻³ mol/1 Tris,pH 8.0, 1% NP-40, and 10% glycerol) containing protease and phosphataseinhibitors, ultrasonicated, and then centrifuged at 12,000×g. Thesupernatants were acidified and neutralized to pH 7.4 before assaying.The tissue levels of GDNF were measured with an ELISA kit (GDNF EmaxImmunoAssay System; Promega, Madison, Wis.), according to the protocolof the supplier. The levels of GDNF were expressed as picograms permilligram of protein. The assay sensitivity ranged from 16 to 1000pg/ml.

Immunohistochemistry.

Muscle sections (10 μm) were fixed in cold acetone, followed byincubation with rabbit anti-FLAG polyclonal antibodies (1:1000; Sigma,St. Louis, Mo.) as primary antibodies and biotinylated anti-rabbitantibodies as secondary ones (1:400; Santa Cruz Bio-technology, SantaCruz, Calif.). Sections were visualized by the avidin-biotin-peroxidasecomplex procedure (Vectastain ABC kits; Vector Laboratories, Burlingame,Calif.) using 3,3-diaminobenzidine as a chromogen.

For double-immunofluorescence staining of muscles, sections weresequentially incubated with blocking solution, polyclonal rabbitanti-FLAG antibodies (1:500; Sigma), FITC-conjugated goat anti-rabbitIgG (1:200; Santa Cruz Biotechnology), andtetramethylrhodamine-conjugated α-bungarotoxin (Molecular Probes,Eugene, Oreg.). Sections were examined and photographed under a confocallaser scanning microscope (TCS NT; Leica, Heidelberg, Germany).

For morphological analysis of the spinal cord, serial transversesections (30 μm) were obtained for Nissl, SMI-32, or CTB immunostaining.Free-floating sections were immunohistochemically stained for SMI-32with a Mouse-on-Mouse kit (M.O.M kit) (Vector Laboratories, Burlingame,Calif.), according to the protocol of the manufacturer. Sectionsprocessed for CTB immunoreactivity were blocked with 5% rabbit serum,followed by incubation with anti-CTB antibodies (1:10000, goat antiserumto CTB; List Biologic). Sections were visualized by standard ABCmethods.

For double immunostaining of the spinal cord, sections were blocked with10% normal goat serum and the blocking solution supplied with the M.O.Mkit for 1 hr, respectively, and then sequentially incubated withpolyclonal rabbit anti-FLAG antibodies (1:250; Sigma) and monoclonalmouse anti-SMI-32 antibodies (1:500) overnight at 4° C. After incubationwith FITC-conjugated goat anti-rabbit IgG (mouse absorbed, 1:200; SantaCruz Biotechnology) and rhodamine-conjugated goat anti-mouse IgG (1:200;Santa Cruz Biotechnology) for 2 hr at room temperature, the sectionswere examined and photographed under confocal laser scanning microscope.

Morphometric analysis and cell counting.

Morphometric analysis was performed on images captured with a CCD camerausing KS 400 image analysis software (Zeiss, Oberkochen, Germany). Themean area of muscle fibers was calculated from counts of >1000 fibers inrandomly selected areas. To compare the number of motoneurons in thespinal cord, neurons were counted in Nissl-stained and SMI-32- andCTB-immunostained sections spanning the cervical and lumbrosacralenlargements in each group, as described previously (Lewis et al., Nat.Genet. (2000) 25:402-405). For each mouse, at least 20 sections in eachsixth serial section were subjected to counting. Only large cellprofiles meeting the following criteria were included: location in theventral horn below a lateral line from the central canal, containing adistinct nucleus with a nucleolus, and possession of at least one thickprocess.

Statistical analyses.

The data were statistically analyzed using repeated-measures ANOVA,followed by a Tukey's honestly significance difference test for multiplecomparisons between groups (StatView 5.0 software; SAS, Cary, N.C.).

B. Results

GDNF Transgene Expression in Muscles of ALS Mice

The amount of GDNF in gastrocnemius muscles was determined by ELISAusing the methods described above. At 110 days of age (7 weeks afterinjection), the GDNF levels in AAV-GDNF vector-treated mice were7985.0±874.0 pg/mg protein, which is >120-fold higher than that in thecontrol ALS group (62.2±20.5 pg/mg protein;p<0.01; n=4). At the time ofdeath, AAV-GDNF vector-treated ALS mice tended to show a decrease inintramuscular GDNF expression (3281.7±667.0 pg/mg protein; n=4). Thereduction of GDNF was assumed to be attributable to the severe atrophyof the transduced muscle fibers in ALS mice, because stable GDNFexpression can last for at least 8 months in age-matched wild-type mice.These data suggested that AAV-GDNF vector could drive substantialtransgenic GDNF expression in ALS mice until the end stage of thedisease.

The pattern of distribution of transgenic GDNF in muscles was examinedby means of immunodetection. FLAG was used as a tag to distinguishtransgene GDNF from its endogenous counterpart. In AAV-GDNFvector-injected mice, strong FLAG immunoreactivity was detected in alarge number of myofibers, both at 110 days of age and at the end stageof the disease. Punctured and reticular staining was observed intransverse sections of muscles, with intense immunoreactivity mainlylocalized in the vicinity of the sarcolemma, indicating thattransgene-derived GDNF was efficiently secreted into the surroundingregions. Substantial FLAG signals could still be detected in atrophiedmyofibers at the end stage of the disease.

Furthermore, double-immunofluorescence staining with anti-FLAGantibodies and α-bungarotoxin was performed. α-Bungarotoxin is amolecular probe that specifically binds to the acetylcholine receptor(AChR) with high affinity on the postsynaptic membranes of NMJs. Theresults showed that more intense immunoreactivity for FLAG wascolocalized with α-bungarotoxin signals, indicating that transgene GDNFwas concentrated primarily in the regions of NMJs. As expected, themuscles treated with AAV-LacZ vector or the vehicle exhibited noimmunostaining for anti-FLAG at any time point.

Preservation of Vector-Treated Muscles

At 110 days of age, the gastrocnemius muscles in the control ALS miceweighed only approximately half those in the age-matched wild-type mice(95.8±19.4 vs 183.0 ±22.2 mg; n=5). However, the gastrocnemius musclesof AAV-GDNF vector-treated ALS mice were approximately 1.68 times(160.1±32.9 mg; p<0.01; n=5) heavier than those of control ALS mice atthe same age.

Histological analysis of muscles in control ALS mice at 110 days of agerevealed widespread groups of small, acutely angulated fibers,consistent with severe neurogenic atrophy. The mean myofiber area wasgreatly decreased (1053.8±581.0 μm²;n=4), being approximately 30% ofthat in age-matched wild-type mice (3517.6±613.5 μm²; n =5). Incontrast, the muscles treated with AAV-GDNF vector showed littleevidence of neurogenic atrophy with a more consistent fiber size, themean myofiber area (2252.8±1035.2 μm²; n=5) reaching approximately 71%of that in wild-type mice and more than two times that in the controlALS group. Additionally, the notable shift of myofibers toward a smallerdiameter observed in control ALS mice was evidently moderated in theAAV-GDNF vector-treated group (FIG. 3), and the percentage of atrophiedmyofibers of <20 μm was significantly decreased (24% in control ALSgroup vs 9% in AAV-GDNF-treated group).

Retrograde Transport of Transgenic GDNF into Spinal Motoneurons

Retrograde axonal transport of GDNF into spinal lumbar motoneurons hasbeen demonstrated in adult rats (Leitner et al., J. Neurosci.(1999)19:9322-9331). Thus, the ability of transgene GDNF to beretrogradely transported to spinal motoneurons in ALS mice was examined.For this purpose, the FLAG tag in transgene GDNF was used to avoidinterference of the results by endogenous GDNF. SMI-32 is a wellcharacterized antibody that specifically recognizes nonphosphorylatedneurofilaments (NP-NFs) and therefore serves as a reliable marker formotoneurons (Carriedo et al., J. Neurosci. (1996) 16:4069-4079). Thus,double immunostaining was performed with SMI-32 and FLAG antibodies onspinal cord sections from ALS mice. At 110 days of age, FLAGimmunosignals could be detected in SMI-32-positive cells in thecorresponding ventral horn in ALS mice at 7 weeks after intramuscularAAV-GDNF vector injection, whereas no FLAG signal was detected in thespinal cords of the control group ALS mice. This was furtherdemonstrated in the subgroup of unilaterally treated ALS mice; FLAGsignals could only be detected in motoneurons of the ventral hornipsilateral to the AAV-GDNF vector-injected side and none in those onthe contralateral AAV-LacZ vector-injected side. AlthoughP-galactosidase signals were widely detected in AAV-LacZ vector-injectedmuscles, they were not observed at all in the corresponding ventral hornof the spinal cord.

As explained above in Example 3, the transgene GDNF that appeared in themotoneurons could have been derived through three possible ways:systemic delivery, retrograde transport of AAV vectors, or retrogradetransport of GDNF fusion protein itself. However, the restricteddistribution and ipsilateral presentation of transgenic GDNF inmotoneurons, as well as its known inability to pass through theblood-brain barrier, exclude the possibility of its systematic deliveryto the spinal cord. To date, most reports show that AAV vectors are notretrogradely transported or are transported in only a very limitedmanner (Chamberlin et al., Brain Res. (1998) 793:169-175; Klein et al.,Exp. Neurol. (1998) 150:183-194; Alisky et al., NeuroReport (2000)11:2669-2673. One recent report, however, has revealed retrogradetransport of an AAV vector itself in the CNS, a reporter greenfluorescent protein being used as a tracer (Kaspar et al., Mol. Ther.(2002) 5:50-56). Thus, the possibility that AAV particles may also havebeen transported to the corresponding motoneurons cannot be completelyruled out. However, the vectors carried to the motoneurons, are assumedto be very limited because no β-galactosidase was detected in thecorresponding spinal motoneurons, despite its wide distribution in thetransduced muscles.

In contrast, the transgenic GDNF was abundantly detected in bothtransduced muscles and the corresponding motoneurons after AAV-GDNFinjection. This finding, combined with the previous reports as well asthe observation regarding β-galactosidase activity, indicates that thetransgenic GDNF in the motoneurons is mainly derived through retrogradeaxonal transport of the GDNF protein. This is consistent with previousstudies (Kordower et al., Science (2000) 290:767-773; Wang et al., GeneTher. (2002) 9:381-389), showing that transgenic GDNF is retrogradelytransported. The finding that transgenic GDNF in muscle fibers waspredominantly accumulated to the regions of NMJs is also compatible withits retrograde transport hypothesized because it is in the axonterminals in which substances secreted from muscle fibers are taken upto be retrogradely transported.

Effect of Transgene GDNF on Spinal Motoneuron Survival

To assess the neuroprotective effect of GDNF on the survival ofmotoneurons, the numbers of spinal motoneurons in the different groupsat 110 days of age were compared. Nissl staining of the spinal cordshowed a severe loss of motoneurons in the ventral horns of the controlALS mice (FIG. 4A). In contrast, in AAV-GDNF vector-treated mice, asignificantly larger number of motoneurons remained in both cervical andlumbar segments (FIG. 4A), suggesting a markedly protective effect ofthe transgenic GDNF on motoneurons.

Staining for NP-NF is a reliable means of assessing the extent ofmotoneuron loss in ALS, in which it has been shown that motoneurondegeneration induces dephosphorylation of NP-NF, resulting in SMI-32staining resistance (Tsang et al., Brain Res. (2000) 861:45-58).Staining was performed on serial sections to evaluate motoneurons withNP-NF. Consistent with the Nissl-staining results, AAV-GDNFvector-treated ALS mice had significantly greater numbers ofSMI-32-positive motoneurons compared with in the control ALS group (FIG.4B). Thus, the motoneuron degeneration, as well as the aberrant NFdephosphorylation in the spinal cord ventral horn of ALS mice, was alsosignificantly inhibited after AAV-GDNF vector administration. Thus, bothSMI-32 staining and Nissl staining confirmed significant rescue ofmotoneurons by AAV-GDNF vector delivery.

In the unilaterally treated subgroup of ALS mice killed at 110 days ofage, many more motoneurons survived in the lumbar spinal cord ventralhorns ipsilateral to the AAV-GDNF vector-injected side than on thecontralateral side treated with AAV-LacZ vector (Nissl staining,17.1±3.2 vs 10.3±1.1; SMI-32-positive neurons, 15.6±1.8 vs 8.8±3.2;p<0.01; n=5). Thus, these findings further suggested that thetherapeutic effect on motoneurons resulted from retrograde transport oftransgenic GDNF on the same side rather than from systemic delivery.

Effect on the Maintenance of Motoneuron Axonalprojections to Muscles

To further quantitatively assess surviving motoneurons that retainedfunctioning neuromuscular projections to the injected muscles, suchmotoneurons were selectively labeled by injection of a neural tracer CTBinto the bilateral gastrocnemius muscles of mice 1 week before beingsacrificed. At 110 days of age, there were much fewer CTB-labeledmotoneurons in control ALS mice than in wild-type mice. However, withAAV-GDNF vector treatment, more CTB-labeled motoneurons were maintainedthan in the control ALS group (20.7±4.9 vs 11.0±2.5%; n=4; p<0.01) (FIG.5A). Transgenic GDNF delivery to muscle thus played an important role inthe maintenance of the axonal projections of corresponding motoneurons.

For more accurate morphometric evaluation of surviving motoneuronslabeled with CTB, we next determined the size and distribution of suchneurons in the lumbar spinal cord. The control group of ALS mice killedat 110 d of age exhibited a significantly smaller mean area ofCTB-labeled neurons than that in age-matched wild-type mice (353.1±173.8vs 733.7±252.0 μm²; n=4; p<0.01), the size distribution being shiftedtoward smaller ones, indicating significant atrophy of CTB-positivemotoneurons (FIG. 5B). In contrast, AAV-GDNF vector treatment of ALSmice markedly decreased the motoneuron atrophy (605.8±248.2 μm² ; n=4;p<0.01 vs control group), and the size distribution shifted towardsmaller ones. Together, these results show that GDNF gene delivery tomuscles can promote the survival and inhibit the atrophy of motoneuronswith axonal projections to target muscles in ALS mice.

Because CTB can be axonally transported to neuronal cell bodies in aretrograde direction and be detected throughout the neuronal cytoplasm(Llewellyn-Smith et al., J. Neurosci. Meth. (2000) 103:83-90), thedetection of CTB-positive motoneurons means that they maintain intactaxonal connection with the AAV-GDNF vector-injected muscles. Thus, CTBlabeling makes it possible to assess the effect of the transgenic GDNFon the spinal motoneurons more accurately than with Nissl or NP-NFstaining alone. This method revealed greater numbers of larger spinalmotoneurons labeled with CTB in AAV-GDNF vector-treated ALS mice. Thesefindings together indicate that intramuscular injection of AAV-GDNFvector can delay the degeneration of motoneurons, thereby allowingprolonged functioning axons in ALS mice.

GDNF Delays the Onset of Disease, Improves Motor Performance, andProlongs Survival in Transgenic ALS Mice

Any group of ALS mice that had AAV-GDNF vector, AAV-LacZ vector, or thevehicle injected in the four limbs at 9 weeks of age showed similarmotor performance, as quantified with a rotarod, until 12 weeks of age.Thereafter, it deteriorated quickly in control ALS mice, whereas theperformance deterioration was significantly delayed in AAV-GDNFvector-treated mice (p<0.05) (FIGS. 6A-6D), indicating significantlyprolonged maintenance of their motor strength. The average age of motordeficit onset in AAV-GDNF vector-treated ALS mice was 114.0±4.0 d(n=12), whereas it was 101.3±5.4 d (n=11) in control ALS mice, thedifference being significant (p<0.01) (FIG. 6A). AAV-GDNF vectortreatment prolonged the mean survival by 16.6±4.1 d compared with in thecontrol ALS mice (138.9±9.2 d in AAV-GDNF vector-treated mice vs122.3±5.7 d in control ALS mice; n=8; p<0.01) (FIG. 6E). These resultsmean that bilateral intramuscular injection of AAV-GDNF vector delayedthe onset of disease by approximately 13% and prolonged the survival oftransgenic ALS mice by approximately 14%.

However, weakness and atrophy of the skeletal muscles, especially in thehindlimbs, ultimately developed in all mice of all groups once motorsymptoms had appeared. The duration of the disease, as evaluated as thenumber of days that elapsed from the onset to the end stage, did notdiffer between the AAV-GDNF vector-treated and control ALS mice(24.0±3.5 vs 21.0±3.5 d;p>0.05). Because GDNF is a secreted protein, weassessed whether the therapeutic benefit of transgene-derived GDNF alsoresulted from systemic circulation after AAV-GDNF vector administrationor not. In a subgroup of unilaterally treated ALS mice that had AAV-GDNFvector injected into their left limbs and AAV-LacZ vector injected intotheir right ones, each mouse moved the AAV-GDNF vector-injected limbsalmost normally until 110 days of age. However, the contralateral limbsdeveloped muscle weakness at as early as 93 days of age, there being awaddling gait. Despite the better motor functions of AAV-GDNFvector-treated limbs, the mice showed no significant difference in therunning time on a rotarod at any speed tested compared with the controlALS mice. The average onset time of motor deficit in this subgroup alsoshowed no significant difference compared with in the control ALS group(102.7±3.1 d; n=7;p>0.05) (FIG. 6D).

To summarize, AAV-GDNF vector-treated ALS mice with four-limbinjections, in contrast to the control ALS group, showed much betterbehavioral performance, with delayed onset of disease and a prolongedlife span, which is in agreement with the attenuation of the motoneuronpathology. In the subgroup with unilateral AAV-GDNF treatment, thetherapeutic effects of GDNF on behavioral and pathological features werelimited to the same treated side, with obvious deterioration of motorperformance on the AAV-LacZ vector-treated side. However, the motorperformance on a rotarod and the onset of disease remained similar tothose in the control group. Thus, it is assumed that the therapeuticbenefit mostly resulted from direct action of transgenic GDNF onmotoneurons after its retrograde transport rather than from the systemicdelivery.

Although bilateral administration of AAV-GDNF vector markedly delayedthe onset of disease and improved the survival of ALS mice, it failed toprolong the length of time from disease onset to death. What is more,despite the substantial expression of GDNF, the AAV-GDNF vector-treatedmice ultimately reached the end stage, when morphological assessmentdemonstrated such severe atrophy of myofibers and massive loss of spinalmotoneurons as in the control ALS mice. It has been reported thatpathological changes occur at asymptomatic stages in ALS mice, andmassive motoneuron death occurs at the end stage (Dal Canto and Gurney,Brain Res. (1995) 676:25-40; Wong et al., Neuron (1995) 14:1105-1116;Mourelatos et al., Proc. Natl. Acad. Sci. USA (1996) 93:5472-5477; Tu etal., Proc. Natl. Acad. Sci. USA (1996) 93:3155-3160; Bruijn et al.,Science (1998) 281:1851-1854; Shibata et al., Acta. Neuropathol. (1998)95:136-142). Thus, the transgene GDNF may exhibit its greatestprotective function for motoneurons in ALS mice at asymptomatic stageswhen the ventral horns have a mild pathology. Once the disease develops,however, GDNF gene therapy may not as readily inhibit the massivemotoneuron death or interfere with the rapidly inevitable progression ofthe disease. In the above experiment, treatment was begun at the age of9 weeks. Administration of GDNF at earlier times and/or together withother neurotrophic factors (Bilak et al., Amyotroph. Lateral Scler.Other Motor Neuron Disord. (2001) 2:83-91) may lead to better results.

The above studies show that intramuscular injection of AAV-GDNF vectorinto the transgenic ALS mice model results in sustained substantialbiosynthesis of GDNF in the muscles and retrograde transport to thecorresponding spinal motoneurons. Expression was sustained until theterminal stage in ALS mice, likely guaranteeing a continual supply ofbiologically synthesized GDNF to the motoneuronal axon terminals in themuscles. Furthermore, this transgene expression not only significantlyprevented the loss of motoneurons but also lead to marked attenuation ofthe manifestation of the disease and prolonged survival of thetransgenic ALS mice. Additionally, AAV-GDNF vector-treated ALS mice, incontrast to the control ALS group, showed much better behavioralperformance.

Accordingly, novel methods for treating motoneuron diseases, such asALS, are provided. Although preferred embodiments of the subjectinvention have been described in some detail, it is understood thatobvious variations can be made without departing from the spirit and thescope of the invention as defined by the appended claims.

1. A method of delivering a recombinant adeno-associated virus (AAV)virion to a muscle cell or muscle tissue of a mammalian subject with amotoneuron disorder, said method comprising: (a) providing a recombinantAAV virion which comprises a polynucleotide encoding a glial cellline-derived neurotrophic factor (GDNF) operably linked to controlelements capable of directing the in vivo transcription and translationof said GDNF; and (b) delivering said recombinant AAV virion directlyinto said muscle cell or muscle tissue of said subject, whereby saidGDNF is expressed at a level which provides a therapeutic effect in saidmammalian subject.
 2. The method of claim 1, wherein said muscle cell ortissue is derived from skeletal muscle.
 3. The method of claim 1,wherein said recombinant AAV virion is introduced into said muscle cellin vivo.
 4. The method of claim 3, wherein said recombinant AAV virionis introduced by intramuscular injection.
 5. The method of claim 1,wherein said recombinant AAV virion is introduced into said muscle cellin vitro.
 6. A method of treating a mammalian subject with a motoneurondisorder comprising administering intramuscularly to said subjectrecombinant adeno-associated virus (AAV) virions comprising apolynucleotide encoding a glial cell line-derived neurotrophic factor(GDNF) polypeptide operably linked to expression control elementscapable of directing the in vivo transcription and translation of saidGDNF to provide a therapeutic effect.
 7. The method of claim 6, whereinthe subject is human and the polynucleotide encodes a human GDNF.
 8. Themethod of claim 7, wherein the polynucleotide encodes humanpre-pro-GDNF.
 9. The method of claim 6, wherein the control elementscomprise a viral promoter.
 10. The method of claim 9, wherein thepromoter is an MLP, CMV, or RSV LTR promoter.
 11. The method of claim 6,wherein muscle cells are transduced in vivo.
 12. The method of claim 11,wherein the recombinant AAV virions are administered into skeletalmuscle of said subject.